Download Attachment 2.2 Sequencing results

Transcript detection of the AHR gene
in the dog
Tara de Jong BSc
April 2012
Supervisor:
Frank van Steenbeek, PhD student, Genetics
Lab support:
Frank Riemers
Manon Vos
Department of Clinical Sciences of Companion Animals
Research Project Veterinary Medicine – Utrecht University
Transcript detection of the AHR gene in the dog
Contents
Summary ............................................................................................................ 3
Introduction ........................................................................................................ 4
Objective ............................................................................................................ 8
Hypotheses ......................................................................................................... 8
Conservation of the AHR gene................................................................................ 9
Materials and methods .......................................................................................... 14
Results ............................................................................................................... 19
Discussion ........................................................................................................... 25
Conclusion........................................................................................................... 26
References .......................................................................................................... 27
Attachment 1.1 RNA isolation protocol .................................................................... 29
Attachment 1.2 cDNA synthesis protocol ................................................................. 31
Attachment 1.3 PCR amplification protocol .............................................................. 32
Attachment 1.4 SAP/ExoI purification protocol ......................................................... 33
Attachment 1.5 Big Dye Terminator Cycle sequencing protocol .................................. 34
Attachment 1.6 Ethanol precipitation protocol .......................................................... 36
Attachment 1.7 qPCR protocol ............................................................................... 37
Attachment 1.8 Western blotting protocol ............................................................... 38
Attachment 2.1 Sequencing results ........................................................................ 41
Attachment 2.2 Sequencing results ........................................................................ 44
Attachment 2.3 Sequencing results ........................................................................ 46
Attachment 2.4 Sequencing results ........................................................................ 51
Attachment 2.5 Genorm results and meltcurves ....................................................... 54
Attachment 2.6 Sequencing results ........................................................................ 56
Attachment 2.7 Sequencing results ........................................................................ 59
2
Transcript detection of the AHR gene in the dog
Summary
Introduction: the Aryl Hydrocarbon Receptor is best known for its function in regulating
xenobiotic metabolism and dioxin toxicity. [1] AHR activation by environmental
contaminants is considered to be an adaptive response, which could decrease the toxicity
of these environmental contaminants. On the other hand, activation of AHR could also
mediate the toxicity of environmental contaminants.
AHR mRNA is expressed in a variety of human tissues. Highest expression is detected in
the placenta and in liver, while in pancreas, heart and lung expression is also relatively
high. [2]
Research has shown that AHR-deficiency in mice resulted in a loss of teratogenesis caused
by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and susceptibility to chemical
carcinogenesis by benzo(a)pyrene. [10] So AHR plays a role in tumour
promotion/initiation, while recent evidence suggests AHR also plays a role in tumour
progression. [3]
On the other hand, AHR deficient mice have a reduced body weight, display reduced
fecundity and are defective in development of both liver and immune system. In these
mice fetal vascular structures are found in the eyes, kidneys and liver. [1]
Objective: to study the conservation of the AHR gene and investigating whether there is
more than just one transcript of the AHR gene in the dog.
Hypotheses: two hypotheses were formulated, namely:
- The AHR gene is highly conserved.
- There are multiple transcripts of the AHR gene in the dog
Conservation: the protein coding transcripts of human, dog, mouse, rat, horse, chicken,
rabbit and zebrafish were compared by blasting them in NCBI Blast. Both nucleotide
sequences and aminoacid sequences were compared. Query coverages ranging from 47%
to 91% were found taking both methods into account.
Materials and Methods: cDNA from liver tissue was used for the detection of other AHR
transcripts by using different primersets to perform PCR, gel electrophoresis and
sequencing. Based on these sequences primers specific for the new transcript were
designed. cDNA from liver, brain tissue, bone tissue, mammary tissue, placenta, pancreas,
adrenal gland, uterus, ovary, kidneys and leucocytes was used to perform PCR, gel
electrophoresis and qPCR to detect the presence of this new transcript (and original
transcript) in these tissues. The original transcript was also sequenced. All sequencing
results were blasted to the dog genome and the annotated original transcript of the AHR
gene. Last, western blotting was performed to see if both the original and new transcript
are translated to proteins in liver cells.
Results: a smaller transcript was detected in our liver cDNA samples. Based on the found
sequences specific primers were designed to confirm the existence of the new transcript. A
part of exon 2 and 7 and exon 3 till 6 are not present in the new transcript. The qPCR data
showed differences between the original and new transcript in certain tissues. The
sequencing results for the original transcript showed that the annotated intron 10 is
coding. The western blotting showed many non-specific bands so more research needs to
be done before these bands can be distinguished from bands of the AHR transcripts.
Discussion: even though the existence of another transcript has definitely been proven
and the presence in many tissues has been detected, the relative level of expression
remains to be measured in the different tissues. The possibility of this transcript
undergoing nonsense mediated decay also needs further research. For a more complete
picture of the conservation of the AHR gene, more species’ nucleotide sequence and
aminoacid sequence could be compared.
Conclusion: the first hypothesis, that the AHR gene is a highly conserved gene, should be
rejected as query coverages ranging from 47% to 91% were found taking both comparing
methods into account. Even though 91% is high, 47% is very low so the AHR gene is not
highly conserved. The second hypothesis, that there are multiple transcripts of the AHR
gene in the dog, should be confirmed based on our sequencing results and qPCR data. The
new transcript probably consists of exon 1, 2, 7, 8, 9 and 10, with a premature stop codon
in exon 7. Furthermore, the original transcript of the dog consists of 11 exons, instead of
the annotated 12 exons.
3
Transcript detection of the AHR gene in the dog
Introduction
The Aryl Hydrocarbon Receptor (AHR) is best known for its function in regulating
xenobiotic metabolism and dioxin toxicity. The role of AHR is not always protective though,
as stimulation of AHR by certain high affinity AHR agonists can lead to cancer,
immunosuppression, liver damage and fetal malformation. [1]
AHR activation by environmental contaminants is considered to be an adaptive response,
which could decrease the toxicity of these environmental contaminants. On the other hand,
activation of AHR could also mediate the toxicity of environmental contaminants. This goes
for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), for example. [2]
According to a phylogenetic survey, AHR arose over 450 million years ago. Functional
orthologs of AHR were detected in several species in various environments, which indicates
the selective advantage of expressing AHR in an environment not yet polluted by chemical
anthropogenic compounds. What might this selective advantage be then? A small hint
comes from the fact that AHR is being expressed in a variety of tissues and at different
phases of vertebrate development which are inconsistent with AHR being expressed only
as a metabolic response to environmental chemicals. So AHR’s function goes beyond just
being a regulator of xenobiotic metabolism. [1]
AHR is expressed in a variety of human tissues as described above. Highest expression is
detected in the placenta and in liver, while in pancreas, heart and lung expression is also
relatively high (Figure 1).
Endogenous ligands of AHR are arachidonic acid metabolites (such as prostaglandinG2 and
lipoxin4A) and heme metabolites (for example bilirubine), although none of these have
proven to be high-affinity ligands. The same goes for nonligand activators of AHR, such as
stress, cAMP and modified low-density lipoprotein, which are factors not proven to be highaffinity activators. [2]
Over 400 exogenous AHR ligands are known, for example environmental pollutants and
dietary compounds. [3]
Fig. 1. An overview of AHR expression in the different tissues. [4]
The following table refers to literature in which the expression of AHR in the concerning
tissue is described in more detail:
4
Transcript detection of the AHR gene in the dog
Tissue
Placenta
Liver
Pancreas
Heart
Lung
Cerebellum
Kidneys
Skin
Literature
[5]
[5]
[5]
[5]
[5]
[6]
[7]
[8]
Table 1.1
You AHR what you eat [9]: AHR’s role in the intestinal immune system
The statement of vegetables being good for your health has been commonly accepted,
while not many people have given a good thought about the underlying mechanisms.
Research has shown that the consumption of cruciferous vegetables, such as broccoli,
cauliflower and cabbage is important in the intestinal and dermal immune system.
Intraepithelial lymphocytes in skin and intestine express high levels of AHR. These
lymphocytes are involved in promoting epithelial repair and limiting bacterial invasion of
epithelial cells via the gut.
In a research investigating the amounts of intraepithelial lymphocytes in AHR deficient
mice, it was found that these lymphocytes were absent in the intestine and numbers were
reduced in the skin. Neither the development of the lymphocytes’ precursor cells, nor the
migration of these cells to the intestine and skin was affected. It was the maintenance of
these cells when they had arrived at their objective site which was affected. In just a few
days intraepithelial lymphocytes disappeared from the intestine and skin.
In agreement with intraepithelial lymphocytes being involved in epithelial repair it was
found that AHR deficient mice were more susceptible to epithelial injury than wild-type
mice, as they showed accelerated illness after being exposed to an epithelial damaging
agent.
Moreover, the AHR deficient mice also show a small increase in intestinal colonization of
the gram-negative genus Bacteroides, which was associated with a reduced expression of
Regllly, an anti-bacterial protein expressed in intraepithelial lymphocytes and epithelial
cells.
Cell-intrinsic AHR signaling originating within the lymphocytes themselves appears to be
necessary for the maintenance of intraepithelial lymphocytes (Figure 2) Yet it is still
unknown which target genes are activated by AHR and how this leads to the maintenance
of intraepithelial lymphocytes in the epithelium of intestine and skin.
5
Transcript detection of the AHR gene in the dog
Fig. 2. Cell-intrinsic AHR signaling in an intraepithelial lymphocyte. Visible is that certain
compounds (in this example indole-3-carbinol) in cruciferous vegetables are converted to
high-affinity AHR ligands after being exposed to stomach acid. After the binding of this
ligand to AHR, translocation to the nucleus occurs and the complex functions as a
transcription factor, activating gene expression. [9]
The evolutional explanation for the ingestion of food being linked to the intestinal immune
defense could be the conservation of energy in times of food scarcity. As the ingestion of
food is accompanied with the ingestion of many likely pathogenic environmental bacteria,
is seems logical the intestinal immune system adjusts.
The question raised by the discovery of consumption of cruciferous vegetables being linked
to your intestinal and dermal immune system is if there are other dietary ligands for AHR.
It seems logical more dietary AHR ligands exist given the variety of most mammalian
diets. It would be remarkable if a part of the intestinal and dermal immune system would
only depend on cruciferous vegetables, but further investigation will tell. [9]
How does AHR operate?
Normally, AHR is located in the
cytoplasm where it forms a complex with
several proteins, like Hsp90, XAP2 and
p23 proteins. [10] The binding of toxic
components, for example polycyclic
aromatic hydrocarbons (PAH’s) such as
benzo(a)pyrene, leads to receptor
dimerization with another protein called
aryl hydrocarbon nuclear transclocator
(ARNT) [1] This heterocomplex
translocates to the nucleus where it now
can bind the xenobiotic responsive
element (XRE) on the DNA. Doing so, the
complex acts as a transcription factor as
it activates gene expression and in this
case for genes encoding phase I and
phase II xenobiotic metabolizing
enzymes (Figure 3). This way, the
organism is protected from exposure to
certain toxic environmental
contaminants.
Fig. 3. Schematic illustration of how AHR
operates. [10]
6
Transcript detection of the AHR gene in the dog
AHR-related factor (Ahrr), which normally is located within the nucleus and bound to
ARNT, competes with AHR. Like AHR it binds to ARNT and this Ahrr/ARNT complex can also
bind the XRE. In contrast with the AHR/ARNT complex, the Ahrr/ARNT complex acts as a
transcriptional repressor. [10]
The same pathway is activated when halogenated aromatic hydrocarbons, such as 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) bind AHR. The heterocomplex again binds the XRE on
the DNA in the promoter region of genes encoding Cytochrome P4501A1 (CYP1A1), 1A2
and 1B1. Unfortunately, the CYP1A1, 1A2 and 1B1 enzymes metabolically activate
procarcinogens to ultimate carcinogens, causing tumour promotion. As AHR also upregulates the expression of DNA polymerase ĸ, which replicates the DNA in an error-prone
manner, this also leads to tumour promotion.
It may not be a surprise that research has shown that AHR-deficiency in mice resulted in a
loss of teratogenesis caused by TCDD and susceptibility to chemical carcinogenesis by
benzo(a)pyrene. [10] On the other hand, mice expressing a continuous active AHR are
more susceptible to stomach tumours. [11]
So AHR plays a role in tumour promotion/initiation, while recent evidence suggests AHR
also plays a role in tumour progression. [3] In adult tissues the inhibition of somatic cell
proliferation is continuously repressed by cell-cell contact. This constantly active
mechanism is also referred to as contact-dependant inhibition of growth. [12]
Cell-cell contact is not only a regulator of proliferation, but also of cell differentiation and
cell motility, thus tumour progression (and also tumour promotion) is triggered by a loss of
cell-cell contact.
Increasing evidence suggests that activation of AHR leads to deregulation of cell-cell
contact and thus promotes tumour progression. [3] For example, AHR expression is higher
in invasive than non-invasive tumour tissue. [13, 14] Also, the level of AHR expression
correlated with malignancy in lung tumours. [15] Moreover, increased invasion and poor
prognosis in human urothelial tumours is associated with AHR expression being
upregulated. [16]
TCDD, a high-affinity AHR ligand, induces a release from contact inhibition in the rat liver
oval cell line WB-F344. [17, 18] As exposure of this cell-line to other AHR ligands, such as
PAH’s also induces a release from contact inhibition, this supports AHR being involved in
tumour initiation and progression. [19-22] The TCDD-dependant release from contact
inhibition seems to be a common phenomenon as it is observed in several cell lines. [23]
In the human breast cancer epithelial cell line MCF-7 a down regulation of E-cadherin (a
transmembrane protein playing an important role in cell-adhesion), loss of cell-cell
adhesion and increased mobility of the cells was found after exposure to TCDD. [24] This
also supports the idea of AHR being involved in tumour progression.
The other side of the story is that AHR deficient mice display reduced fecundity and are
defective in development of both liver and immune system. The cause of the reduced
hepatocyte size turned out to be portosystemic shunting due to a patent ductus venosus.
[1] Fetal hepatic necrosis caused by compromised perfusion also contributed to the liver
defect in AHR deficient mice. [2]
Other fetal vascular structures were found in the adult mice, for example a persistent
arteria hyaloidea in both eyes and an immature sinusoidal structure in the kidneys. This
has led to the idea that AHR functions as a regulator of genes encoding proteins which are
involved in the remodeling of fetal vascular structures.
Not all fetal structures are persistent in AHR-deficient mice though, as the ductus
arteriosus was closed normally. [1]
7
Transcript detection of the AHR gene in the dog
Objective
The objective of my research is to study the conservation of the AHR gene and to find out
whether there is more than just one transcript of the AHR gene in the dog. The original
transcript of the dog will also be sequenced and these results will be compared to the
annotated data.
Hypotheses
The first hypothesis related to the outcome of this research is:
The AHR gene is highly conserved.
The second hypothesis is:
There are multiple transcripts of the AHR gene in the dog.
8
Transcript detection of the AHR gene in the dog
Conservation of the AHR gene
Comparing the different species’ nucleotide sequence
In human, five transcripts of AHR are known (Figure 4) Three of these transcripts are
noncoding transcripts that do not contain an open reading frame. One transcript is thought
to undergo nonsense mediated decay, a process which detects nonsense mutations and
prevents the expression of truncated or erroneous proteins.
The protein coding human transcript consists of 2547 basepairs (without the untranslated
region or UTR) and consists of 11 exons.
Fig. 4. Overview of the five transcripts of the AHR gene in humans. [25]
In the dog, only one transcript of the AHR gene is annotated (Figure 5) This transcript is a
protein coding transcript which consists of 2535 basepairs (without the UTR) and is thus
about the same size compared to human transcript. This transcript consists of 12 exons.
Fig. 5. The one known protein coding transcript of the AHR gene in the dog. [25]
In mice, four transcripts of the AHR gene are known (Figure 6) One of these is a noncoding
transcript containing intronic sequence. Another transcript is thought to undergo nonsense
mediated decay. The other two transcripts are protein coding transcripts, one consists of
2649 basepairs (without UTR) and 13 exons, while the other consists of 2418 basepairs
(without UTR) and 11 exons.
Fig. 6. The four known transcripts of the AHR gene in the mouse. [25]
In the rat, two transcripts of the AHR gene are known (Figure 7) Both these transcripts are
protein coding. One of the transcripts consists of 2562 basepairs (without UTR) and 11
exons, while the other consists of 1857 basepairs (without UTR) and 7 exons. [25]
9
Transcript detection of the AHR gene in the dog
Fig. 7. The two known transcripts of the AHR gene in the rat. [25]
In horses, one transcript of the AHR gene is known (Figure 8) This transcript is protein
coding and consists of 1887 basepairs (without the UTR) and 11 exons.
Fig. 8. The protein coding transcript in the horse. [25]
In chickens, one transcript of the AHR gene is known (Figure 9) This transcript is protein
coding and consists of 2520 basepairs (without the UTR) and 10 exons.
Fig. 9. The protein coding transcript in the chicken. [25]
In rabbits, one transcript of the AHR gene is known (Figure 10) This transcript is protein
coding and consists of 2544 basepairs (without the UTR) and 11 exons.
Fig. 10. The protein coding transcript in the rabbit. [25]
In zebrafish, three protein coding transcrips are known (Figure 11) One of these
transcripts consists of 2427 basepairs (without the UTR) and 12 exons, while the other
transcripts consist of 2424 basepairs, 11 exons and 2373 basepairs and 10 exons
respectively.
Fig. 11. The protein coding transcripts in zebrafish. [25]
10
Transcript detection of the AHR gene in the dog
For comparing the protein coding transcripts between different species to estimate the
degree of conservation of the AHR gene, NCIBI’s align two sequences was used.
All species’ protein coding transcripts were compared:
Human
Dog
Mouse
Rat
Horse
Chicken
Rabbit
vs
vs
vs
vs
vs
vs
vs
dog, mouse, rat, horse, chicken, rabbit and zebrafish
mouse, rat, horse, chicken, rabbit and zebrafish
rat, horse, chicken, rabbit and zebrafish
horse, chicken, rabbit and zebrafish
chicken, rabbit and zebrafish
rabbit and zebrafish
zebrafish
When the protein coding human transcript was compared to the canine transcript a query
coverage of 87% was found. This means 87% of the human transcript shows similarities
with the canine transcript. Almost the entire canine transcript showed similarities with the
human transcript, so almost all 2535 basepairs were found in the human transcript, except
for 30 gaps here and there.
When the protein coding human transcript was compared to the protein coding transcripts
in the mouse a query coverage of 77% was found when compared to the biggest and
smallest transcript.
When the human transcript was compared to the protein coding transcripts in the rat
query coverages of 76% and 70% were found when compared to the biggest and smallest
transcript respectively.
Furthermore, query coverages of 86%, 70% and 82% were found when the human
transcript was compared to the equine transcript and the protein coding transcripts in
chicken and rabbit respectively.
Last, the human transcript was compared to the three protein coding transcripts in
zebrafish. Interestingly, a query coverage of 69% was found for all three comparisons
Overall, fairly high percentages are observed when the human transcript is compared to
the other transcripts.
When the canine transcript was compared to both protein coding transcripts of the mouse
a query coverage of 76% was found when compared to the biggest and smallest transcript.
When the canine transcript was compared to both protein coding transcripts in the rat
query coverages of 76% and 71% were found when compared to the biggest and smallest
transcript respectively.
Query coverages of 88%, 71%, and 81% were found when the canine transcript was
compared to the equine transcript and the transcripts in chicken and rabbit respectively.
Last, the canine transcript was compared to the three protein coding transcripts in
zebrafish. A query coverage of 67% was found for all three comparisons. For a small
overview of the query coverages found by comparing all species’ protein coding transcript I
refer to table 1.2
Species
Hm
Dg
M1
M2
R1
R2
Hr
Ch
Rb
Zf1
Zf2
Zf3
Bp
2547
2535
2649
2418
2562
1857
1887
2520
2544
2427
2424
2373
Hm
x
87
77
77
76
70
86
70
82
69
69
69
Dg
87
x
76
76
76
71
88
71
81
67
67
67
M1
77
76
x
x
89
87
74
74
77
68
68
68
M2
77
76
x
x
90
87
75
74
77
68
68
68
R1
76
76
89
90
x
x
75
74
77
67
67
67
Table 1.2
11
R2
70
71
87
87
x
x
71
70
72
68
68
68
Hr
86
88
74
75
75
71
x
73
79
70
70
70
Ch
70
71
74
74
74
70
73
x
73
69
69
69
Rb
82
81
77
77
77
72
79
73
x
69
69
69
Zf1
69
67
68
68
67
68
70
69
69
x
x
x
Zf2
69
67
68
68
67
68
70
69
69
x
x
x
Zf3
69
67
68
68
67
68
70
69
69
x
x
x
Transcript detection of the AHR gene in the dog
Inscription:
Bp = basepairs (without UTR)
Hm = human
Dg = dog
M1 = mouse 1
M2 = mouse 2
R1 = rat 1
R2 = rat 2
Hr = horse
Ch = chicken
Rb = rabbit
Zf1 = zebra fish 1
Zf2 = zebra fish 2
Zf3 = zebra fish 3
The query coverages found range from quite low to fairly high, as 70% is considered quite
low, while 90% fairly high. For a small overview of the query coverages found I refer to
table 1.2 Mouse 1 and rat 1 refer to the biggest protein coding transcript in these species,
whereas the number 2 refers to the smallest protein coding transcript in these species. The
same goes for the zebrafish, 1 refers to the biggest, while 3 refers to the smallest protein
coding transcript.
Comparing the different species’ aminoacids
Another method to estimate the degree of conservation of the AHR gene is to compare the
different species’ aminoacid sequence. Again, the protein coding transcripts of human,
dog, mouse, rat, horse, chicken, rabbit and zebrafish were compared.
The human protein coding transcript consists of 848 aminoacids. When the human
transcript was compared to the canine aminoacid sequence a query coverage of 84% was
found.
Query coverages of 71% and 72% were found when the human transcript was compared
to the biggest and smallest transcript of the mouse respectively.
When compared to the biggest and smallest transcript of the rat query coverages of 72%
and 57% were found respectively.
When compared to the transcripts of horse, chicken and rabbit query coverages of 80%,
64% and 78% were found.
Last, the human transcript was compared to the biggest, middle and smallest transcript of
zebrafish. A query coverage of 52% was found for all three comparisons.
The canine protein coding transcript consists of 844 aminoacids. A query coverage of 70%
was found when compared to the biggest and smallest transcript of the mouse.
When compared to the biggest and smallest transcript in the rat query coverages of 72%
and 58% were found respectively.
Query coverages of 83%, 66% and 76% were found when compared to the transcripts of
horse, chicken and rabbit.
Last, the canine aminoacid sequence was compared to the three transcripts in zebrafish. A
query coverage of 52% was found for all three comparisons. For a small overview of the
query coverages found by comparing all different species’ aminoacid sequences I refer to
table 1.3.
Species
Hm
Dg
M1
M2
R1
R2
Hr
Ch
Rb
Zf1
Zf2
Zf3
Table 1.3
AA
848
844
882
805
853
618
628
839
847
808
807
790
Hm
x
84
71
72
72
57
80
64
78
52
52
52
Dg
84
x
70
70
72
58
83
66
76
52
52
52
M1
71
70
x
x
90
84
66
60
70
50
50
51
M2
72
70
x
x
91
85
67
62
71
50
50
51
R1
72
72
90
91
x
x
67
61
70
49
49
49
12
R2
57
58
84
85
x
x
58
46
55
58
58
58
Hr
80
83
66
67
67
58
x
63
73
47
47
47
Ch
64
66
60
62
61
46
63
x
63
52
52
52
Rb
78
76
70
71
70
55
73
63
x
51
51
51
Zf1
52
52
50
50
49
58
47
52
51
x
x
x
Zf2
52
52
50
50
49
58
47
52
51
x
x
x
Zf3
52
52
51
51
49
58
47
52
51
x
x
x
Transcript detection of the AHR gene in the dog
Inscription:
R2 = rat 2
Hr = horse
Ch = chicken
Rb = rabbit
Zf1 = zebra fish 1
Zf2 = zebra fish 2
Zf3 = zebra fish 3
AA = Amino-acids
Hm = human
Dg = dog
M1 = mouse 1
M2 = mouse 2
R1 = rat 1
The query coverages found range from low to fairly high (46%-91%) Interestingly, nearly all query
coverages found by comparing the different species’ aminoacid sequences are lower than the query
coverages found by comparing the different species’ basepairs. The only exception is when the
biggest transcript of the rat is compared to both transcripts in the mouse. But, just a difference of
1% is found between these query coverages. An explanation could be due to the fact that when the
first or second basepair of a codon changes, the whole aminoacid changes. When basepairs are
compared, one differing basepair at the beginning of a codon would still lead to a query coverage
of 66,7%, while no similarity would be found if aminoacids would be compared.
In many cases the query coverages found by comparing the different species’ aminoacid sequences
differed more than 10% compared to the query coverages found by comparing the different
species’ basepairs. This goes for all comparisons made with zebrafish and most comparisons with
the smallest transcript in the rat and chicken.
As the aminoacids ultimately determine the shape of the protein more value should be given to the
query coverages found by comparing the different species’ aminoacid sequences. Based on the
aminoacid sequences human, horse and dog are much alike, the same goes for mouse and rat. Low
query coverages were found for all comparisons including the three transcripts of zebrafish. The
same goes for the chicken. Including the transcript of the rabbit two fairly high query coverages
were found (compared to humans and the dog), while the other query coverages found are quite
low.
Based on these results the conclusion could be drawn that the AHR gene is not very conserved.
13
Transcript detection of the AHR gene in the dog
Materials and methods
Experimental design
6 liver samples were used for performing RNA isolation and cDNA synthesis. This cDNA together
with cDNA from 6 other liver samples were used for the detection of other transcripts of the AHR
gene. Primers were ordered specific for each exon of the original transcript. These primers were
used in different combinations to find out whether transcript differences could be detected and if so
out of which exons it consists.
The cDNA liver samples combined with different primersets were used for amplification, gel
electrophoresis and if a product was visible the Big Dye Terminator Cycle Sequencing was
performed to find the exact sequences of these bands.
Before sequencing PCR products were diluted or a SAP/ExoI purification was performed. The
sequences were blasted in NCBI to the dog genome to check whether the used primersets were
specific enough and thus matched AHR. This way, the sequence of the original transcript was found
and compared to the sequence on NCBI. Sequences from another transcript was also detected and
compared to the original transcript.
cDNA from other tissues was used to see whether this new transcript and original transcript could
be detected in these tissues. cDNA from tissues of different parts of the brain, bone tissue,
mammary tissue, placenta, pancreas, adrenal gland, uterus, ovary, kidneys and leucocytes was
used to perform PCR and for most tissue samples also qPCR.
Last, Western blotting was performed to see if both the original and new transcript could be made
visible and are thus translated to proteins in liver tissue.
Tissue samples
12 Liver samples from healthy GDL beagles were used during this research. These samples were
named 4,5 and 8-17.
cDNA from different parts of the brain was used for the detection of the AHR transcripts. 19 brain
samples were used in total, which consisted of 10 cortex samples (2 samples from the cranial lobus
temporalis, the lobus occipitalis, the lobus frontalis, the lobus parietalis and from the middle lobus
temporalis) and 2 samples from the gyrus dentatus (hippocampus), 2 samples from the thalamus,
2 samples from the cerebellum, 2 samples from the ammon’s horn (hippocampus) and 1 sample
from the hypothalamus.
All brain samples originate from healthy labradors or beagles.
Besides liver and brain samples cDNA from many other tissues were used also. cDNA from the
following (normal) tissues was used: pancreas, adrenal gland, uterus, ovary, kidneys, leucocytes,
placenta, mammary tissue (2 samples), bladder tissue, colonic tissue, cervical tissue and bone
tissue (2 samples) The bone tissue came from a Miniature Poodle and a Great Dane.
15 liver protein samples were used to perform Western blotting. 6 of these samples came from
healthy beagle dogs, 6 from Irish Wolfhounds (intrahepatic shunt cases), 2 from Cairn terriers
(extrahepatic shunt cases) and 1 from a Golden retriever (intrahepatic shunt case)
RNA isolation
RNA isolation was performed using a portion of frozen liver tissue. Disruption was performed using
a mortar and pestle followed by homogenization using a QIAshredder homogenizer. A volume of
350ųl buffer RLT was used in step 3 of the protocol (see attachment 1.1)
In step 5 1 volume of 50% ethanol was added to the cleared lysate.
After step 6 of the protocol, the optional on-column DNase digestion was performed with the
RNase-Free DNase Set. This way elimination of genomic DNA contamination was guaranteed.
Instead of 10ųl DNase stock solution and 70ųl buffer RDD in step D2 of the optional on-column
DNase digestion procedure 45ųl of DNAase stock solution and 315ųl of buffer RDD was used.
The samples were eluated in 50ųl RNase-free water.
The protocol used was from the Qiagen RNeasy® Mini Handbook Fourth Edition. After collection
concentration of RNA was measured with the Nanodrop.
14
Transcript detection of the AHR gene in the dog
cDNA synthesis
After RNA isolation cDNA synthesis was performed using 10µl of RNA template, containing 0.5µg
RNA. For the exact protocol I refer to attachment 1.2.
Primers
Before a PCR would be performed, the right annealing temperature would be determined via a
gradient between 55°C and 65°C. The right annealing temperature for the primersets 1 forward, 9
reverse; 2 forward, 5 reverse and 2 forward, 9 reverse was 58°C.
All the forward primers were based on the sequence at the beginning of the relative exon, while all
the reverse primers were based on the sequence at the end of the relative exon.
A gradient was also run on primerset break4 forward, 9 reverse. The right annealing temperature
for this primerset was 55°C.
61°C was the right annealing temperature for the RPS19 and 2 forward, 3 reverse primerset.
The following table displays the sequences of the primers used during this research:
Primer
1 forward
2 forward
6 forward
10 forward
Break1 forward
Break2 forward
Break3 forward
Break4 forward
3 reverse
5 reverse
9 reverse
10 reverse
11 reverse
RPS19 forward
RPS19 reverse
RPS5 forward
RPS5 reverse
GAPDH forward
GAPDH reverse
GUSB forward
GUSB reverse
HNRPH forward
HNRPH reverse
B2M forward
B2M reverse
Sequence
5’–ATC ACC TAC GCC AGC CGC AA-3’
5’-CCA GCT GAA GGA ATC AAG TC-3’
5’-GGT GTC TGC TGG ATA ATT CG-3’
5’-GGC TCC TTC CTG ACA ATA GA-3’
5’-TTC CAA GCG ACA TAG AGA TCC GAA C-3’
5’-TCC AAG CGA CAT AGA GAT CCG AAC C-3’
5’-CCA AGC GAC ATA GAG ATC CGA ACC A-3’
5’-CGA CAT AGA GAT CCG AAC CA-3’
5’-TTG CAG ATG CAG ACC TTC TC-3’
5’-TCA CCT CGG TCT TCA GTA TG-3’
5’-TCA TGC CAC TCT CTC CTG TC-3’
5’-TTC TAT TGT CAG GAA GGA GC-3’
5’-CAT GGC ATA GCA TCA ATC TC-3’
5’-CCT TCC TCA AAA AGT CTG GG-3’
5’-GTT CTC ATC GTA GGG AGC AAG-3’
5’-TCA CTG GTG AGA ACC CCC T-3’
5’- CCT GAT TCA CAC GGC GTA G-3’
5’-TGT CCC CAC CCC CAA TGT ATC-3’
5’-CTC CGA TGC CTG CTT CAC TAC CTT-3’
5’-AGA CGC TTC CAA GTA CCC C-3’
5’- AGG TGT GGT GTA GAG GAG CAC-3’
5’-CTC ACT ATG ATC CAC CAC G-3’
5’-TAG CCT CCA TAA CCT CCA C-3’
5’- TCC TCA TCC TCC TCG CT-3’
5’- TTC TCT GCT GGG TGT CG-3’
Table 1.4
PCR amplification
PCR amplifications were performed in a Gene Amp PCR-system 9700 or a MJ Research Dyad
Disciple Peltier Thermal Cycler. For the exact protocol I refer to attachment 1.3.
Gel electrophoresis
After amplification with PCR, the presence and size of the products were confirmed by gel
electrophoresis. 1.5% agarose gels were used, containing 0.04ųl of ethidium bromide per ml 0.5 x
TBE or 1 x TAE. Each time 10ųl of the PCR products was used with a 2ųl of loading buffer.
For estimating the size of the products, a 10ųl of Promega 100-bp molecular size standard ladder
and/or a 1 Kb molecular size standard ladder was used also. Gel electrophoresis was run in a Bio-
15
Transcript detection of the AHR gene in the dog
Rad Power Pac 300 performing under 80-90V in a buffer of 0.5 x TBE or 1 x TAE. The gels were
visualized under UV light.
If bands were to be excised from the gel, 20ųl of mQ would be added, this would be centrifuged
stored in the refrigerator for a day or two.
SAP/ExoI purification
SAP/ExoI purifications were run in a Gene Amp PCR-system 9700 or a MJ Research Dyad Disciple
Peltier Thermal Cycler. For the exact protocol I refer to attachment 1.4.
Big Dye Terminator Cycle Sequencing
The Big Dye Terminator Cycle was run in a Gene Amp PCR-System 9700 or a MJ Research Dyad
Disciple Peltier Thermal Cycler. Sequencing itself was run in a genetic analyzer 3130xl. The
programs used for collecting and examining the data are Sequencing Analysis 5.2 and DNAstar
LaserGene 8 Seqman.
For the exact protocol I refer to attachment 1.5.
Ethanol precipitation
Instead of performing Sephadex purification previous to sequencing ethanol precipitation could be
performed. For the exact protocol I refer to attachment 1.6.
qPCR
qPCR was run in a Bio-Rad MyiQ2 Icycler. Genorm was used for the ultimate selection of the
reference genes. For the exact protocol I refer to attachment 1.7.
Western blotting
Western blotting was performed using a precast Tris-HCL gel (15%). All samples were diluted to a
concentration of 2 mg/ml. 10 ųl of each sample was used. Non-fat dry milk was used in our
blocking solution. A shaker was used in the washing steps. For the exact protocol I refer to
attachment 1.8.
Experiments
PCR followed by nested PCR
At first PCR with an elongation time of 1.5 min, gel electrophoresis and the big dye terminator
cycle sequencing was performed using the primerset 1 forward, 9 reverse with a total expected
size of 800-900 bp on six liver samples. This way the original transcript could be sequenced and
possibly another transcript could be detected.
After gel electrophoresis 8 smaller bands compared to the original transcript were found indeed and
thus excised and used performing a nested PCR with a shorter elongation time of 20s. In this PCR
the primerset 2 forward, 5 reverse was used. One of these PCR products was then used in
performing the big dye terminator cycle sequencing.
2 forward, 9 reverse
The next step was to try the primerset 2 forward, 9 reverse on six liver cDNA samples performing
PCR (elongation time 20s), gel electrophoresis and the big dye terminator cycle sequencing.
After gel electrophoresis the samples 4, 5, 8 and 9 showed two bands, the bigger one is probably
the original transcript and the small band the smaller transcript we are trying to detect. These
smaller bands were excised from the gel so they could be sequenced. Based on these sequences
break primers were designed.
Break primers
As mentioned earlier, the break primers sequences were based on the different overlap between
exon 2 and 7 we found using the 2 forward, 9 reverse primerset. As only the primerset break4
16
Transcript detection of the AHR gene in the dog
forward, 9 reverse gave good sequencing results the other primersets will not be mentioned
further. 8 Liver samples were used.
The break4 forward primer was thus based on the sequence of sample 4, primerset 2 forward, 9
reverse and binds right before and the overlap of exon 2 and 7 itself.
As the break4 forward, 9 reverse primerset produced the same sequence (with the same overlap
between exon 2 and 7) as it was based on, this primerset was tried on all 12 samples. Again, PCR
(elongation time 15s), gel electrophoresis, SAP/ExoI purification and the terminator cycle
sequencing were performed.
As the same overlap between exon 2 and 7 was found in 10 samples, the next step was to try this
primerset on cDNA samples from other tissues to see if we could find the same sequence (which is
probably an AHR transcript) in other tissue besides liver tissue.
Other tissues
As described before cDNA from brain tissue, pancreas, adrenal gland, uterus, ovary, kidneys,
leucocytes, placenta, mammary tissue and bone tissue was used to perform PCR (elongation time
15s) and gel electrophoresis. The primersets used were the break4 forward, 9 reverse primerset
(for detection of the small AHR transcript), 2 forward, 3 reverse primerset (for detection of the
original transcript) and a RPS19 primerset (to check the reaction) on all 30 cDNA samples.
Exon 10 and 11 of the original transcript
Besides detecting other transcripts than the original transcript, the original transcript was
sequenced. A part of the original transcript had already been sequenced by using the primerset 1
forward, 9 reverse.
As mentioned earlier, the original transcript of the dog exists out of 12 exons, while the biggest
human transcript exists out of 11 exons. When blasting the original transcript of the dog to the
biggest human transcript the first 9 exons of the dog are very similar to the first 9 exons of the
human transcript.
When blasting exon 10 of the dog (696 basepairs) to the human exon 10 (1243 basepairs) exon 10
of the dog is very similar to the first part of the human exon 10, while exon 11 (535 basepairs) of
the dog is very similar to the second part of the human exon 10.
When blasting exon 10 and 11 of dog to the human exon 10 at once in NCBI the following graphic
is showed:
Fig. 12. Exon 10 and 11 of the dog blasted to the human exon 10. [21]
Moreover, taking a look at the intron between exon 10 and 11 in the dog, the length of this intron
is very short (27 basepairs), while the sequence does not start with GT and does not end with AG
like other introns. Also interesting is the CAG repeat in this intron, which is not present in the
human transcript.
- Intron between exon 10 and 11 in the dog: acagcagcagcagcagcagcagcagct
This all has led to the idea of this intron not being an intron and thus exon 10 and 11 in the dog
being just one exon.
Therefore primers were designed to sequence exon 10 and 11 in the dog in six liver cDNA samples.
PCR (elongation time 80s), gel electrophoresis and the terminator cycle sequencing were
performed using the 10 forward, 11 reverse primerset.
As the PCR product amplified via these primers is quite small, another PCR was performed using
the 6 forward, 11 reverse primerset. This way the possibility of sequencing a piece of gDNA is
excluded. The terminator cycle sequencing would then be performed using the 10 forward, 11
reverse primerset.
17
Transcript detection of the AHR gene in the dog
When blasting exon 12 (147 basepairs) of the dog to the human exon 11 (144 basepairs without
the UTR), a query coverage of 91% is found, so exon 12 of the canine transcript is very similar to
exon 11 of the human transcript.
qPCR and reference genes
qPCR was performed to validate the existence of the new transcript by estimating the relative level
of expression for both the original and new transcript (using the 2 forward, 3 reverse and break4
forward, 9 reverse primerset respectively) in several tissues. 16 Samples were used, which
included cDNA from pancreas, adrenal gland, uterus, ovary, kidneys, leucocytes, mammary tissue
(2 samples), bladder, colonic tissue, cervical tissue, liver tissue (3 samples) and 2 negative control
samples.
qPCR was performed for 6 reference genes to create a reliable reference gene set, which was used
for normalizing the results from the genes of interest. qPCR plates were run to acquire the values
of the following reference genes: GAPDH, GUSB, HNRPH, B2M, RPS19 and RPS 5. Genorm was
used to rank the stability of the reference genes.
Length new transcript
Besides measuring the relative expression of the new transcript in different tissues, thereby
validating the existence of the new transcript and determining in which tissues this transcript is
present, the total length of the new transcript was also determined by performing PCR using
different primersets.
PCR (elongation time 80s), gel electrophoresis and the terminator cycle sequencing were
performed using the break4 forward, 10 reverse primerset.
The break4 forward primer was also combined with a 11 reverse, 12 reverse and UTR reverse
primer but no bands could be visualized after PCR and gel electrophoresis were performed.
Confirmation new transcript
The break1 forward, break2 forward and break3 forward were designed as an extra confirmation
for the existence of the new transcript. These primers are based on the same sequence as the
break4 forward primer was based on, but consist of more basepairs compared to the break4
forward primer and are thus more specific. This way, the possibility of our results being based on a
improperly working primer is prevented.
All three forward primers were combined with the 9 reverse primer. PCR (elongation time 30s), gel
electrophoresis and the terminator cycle sequencing were performed using 6 cDNA liver samples
for each primerset.
Western blotting
Taking a more thorough look at the new transcript shows a premature stop codon is present in
exon 7. This had led to the idea that this transcript might undergo nonsense mediated decay and is
thus not translated to protein but degraded. So Western blotting was performed as a control if our
original transcript and our new transcript could be made visible and are thus translated to proteins
in liver cells. The original transcript has a weight of 96kD, while if the new transcript would be
translated it would have a weight of 12kD (150 basepairs/ 50 aminoacids)
As primary antibody a polyclonal Anti-AHR antibody from a rabbit was used. Our secondary
antibody was an anti-rabbit antibody. As our control a monoclonal primary tubulin antibody from a
mouse was used (binds to an canine tubulin epitope, as well as other species’ tubulin epitopes),
while an anti-mouse IgG antibody was used as secondary antibody.
18
Transcript detection of the AHR gene in the dog
Results
RNA isolation
Liver sample
RNA concentration
ng/µl
98.9
272.5
98.6
54.0
162.0
284.3
4
5
8
9
10
11
Degree of
contamination
2.13
2.06
2.11
2.12
2.09
2.06
Remains of ethanol
1.77
2.19
1.68
1.49
2.19
1.99
Table 1.5
PCR followed by nested PCR
Each PCR product could be visualized on gel after gel electrophoresis, except our negative control,
which showed no band as to expect. Moreover each sample showed at least two bands using the
primerset 1 forward, 9 reverse. A bigger band of approximately 800-900 basepairs and a smaller
band of about 300 basepairs was visible, which may be the smaller transcript we were trying to
detect (Figure 13) In sample 4 and 11 we even found a third faint band of 100-200 basepairs.
Unfortunately, these two faint bands are not visible anymore in figure 13.
A part (up to exon 9) of the original transcript could be sequenced (for the exact sequences, see
attachment 2.1) after the first PCR. In attachment 2.1, the 3 samples (sample 4, 8 and 11)
showing the best sequencing results are displayed.
After the nested PCR just one band could be visualized after gel electrophoresis (Figure 14) This
band was from the excised band of 300 basepairs, sample 8. After sequencing only the 2 forward
primer showed results. This could mean the small transcript consists out of at least exon 2, but
probably not exon 5. For the exact sequence, see attachment 2.1.
1 kb ladder
1 kb ladder
4
5
8
9
10
11
nc
4
Fig. 13. On the left is the 1 kb ladder
visible, then our six liver samples (from 4
to 11) and last our negative control.
100 bp ladder
5
8
9
10
11
4
11
Fig. 14. On the left again our 1 kb
ladder, then our excised bands (first the
six 300 basepairs bands, then the two
100-200 basepairs bands) and to the
total right side a 100 basepairs ladder.
2 forward, 9 reverse
In Figure 15 are the results of the gel electrophoresis visible. Only sample 4, 5, 8 and 9
show a smaller band besides the prominent bigger band.
The smaller bands from sample 4, 5, 8 and 9 were sequenced. (for the exact sequences
see attachment 2.2) Interestingly, the overlap between exon 2 and 7 differs between the
found sequences. This could be due to difference in splicing, or perhaps the primers are
not specific enough.
19
Transcript detection of the AHR gene in the dog
4
5
8
9
10
11
Samples
1 kb ladder
100 basepairs ladder
Fig. 15. On the left is a 1 kb ladder visible, then our six liver samples (4 to 11), then a
100 basepairs ladder. The smaller bands which were excised from the gel so they could be
sequenced are outlined in this figure.
Break primers
After gel electrophoresis all 12 samples showed one band of approximately 100-200
basepairs (Figure 16 and Figure 17) 10 samples showed sequencing results which
confirmed the existence of a smaller transcript. For the exact sequences of these bands I
refer to attachment 2.3.
100 bp
4
5
8
9
10
11
100 bp 12 13
Fig. 16. All six liver samples showed one
band after PCR with primerset break4
forward, 9 reverse was performed.
14
15
16
17
8
9
10
11 nc
100 bp
Fig. 17. All 10 samples showed the same
band after PCR with the break4 forward,
9 reverse primerset. Our negative control
is negative.
Other tissues
After gel electrophoresis all 30 samples showed bands the right size for all 3 primersets.
To illustrate this see Figure 18, 19 and 20 where the PCR products from pancreas, adrenal
gland, uterus, ovary, kidneys, leucocytes, bone tissue, placenta, hypothalamus and
mammary tissue are visualized. Interestingly, if these bands happen to be transcripts of
the AHR gene, this means mammary tissue also expresses AHR which is in contrast with
Figure 1.
20
Transcript detection of the AHR gene in the dog
100 bp ladder
pan
ag
ut
ov
kid
leu
100 bp ladder
bmp
bgd
Fig. 18. The first three bands are samples from pancreas (pan), primerset RPS19; 2
forward, 3 reverse and break4 forward, 9 reverse respectively (this goes for every tissue),
the second three bands from adrenal gland (ag), then uterus (ut), ovary (ov), kidneys
(kid), leucocytes (leu), bone tissue Miniature Poodle (bmp) and bone tissue Great Dane
(bgd).
100 bp ladder
100 bp ladder
mammary tissue samples
hypothalamus
Fig. 19. 2 samples of mammary tissue
(primersets RPS19; 2 forward, 3 reverse
and break4 forward, 9 reverse)
placenta
Fig. 20. The first three bands are
samples from hypothalamus and the last
three bands from placental tissue
(primerset RPS19; 2 forward, 3 reverse
and break4 forward, 9 reverse
respectively)
Exon 10 and 11 of the original transcript
After gel electrophoresis four samples showed a band for the 10 forward, 11 reverse primerset
(Figure 21) Sample 4 showed a very faint band, while sample 10 did not show any bands at all. So
sample 5, 8, 9 and 11 were used to perform sequencing analysis. Unfortunately, all the 11 reverse
sequences failed, but the 10 forward sequences showed the ‘intron’.
The gel electrophoresis results for the 6 forward, 11 reverse primerset are visible in Figure 22.
Only sample 16 did not show a band and was thus not used for the terminator cycle sequencing.
For the exact sequencing results I refer to attachment 2.4.
21
Transcript detection of the AHR gene in the dog
100 bp 4
5
8
9
10
11
100 bp 12
13
14
15
16
17
Fig. 22. 5 samples showed a band after PCR
using the 6 forward, 11 reverse primerset.
Fig. 21. 4 samples showed a clearly visible
band after PCR using the 10 forward, 11
reverse primerset.
qPCR and reference genes
Genorm ranks the reference genes; the most stable reference gene is the best reference gene to
use. For the exact Genorm results and meltcurves for both the original and new transcript see
attachment 2.5.
Based on these results the optimal number of control genes for normalization should be 4 or 5
taking into account that RPS5 and RPS19 influence each other’s expression. The best reference
genes to use (ranked in order of stability) should be RPS19 (and/or RPS5), HNRPH, GUSB and
GAPDH. Table 1.6 shows the average level of expression of all 5 reference genes together and the
relative level of expression of both the original and new transcript.
Tissue
Pancreas
Adrenal
gland
Uterus
Ovary
Kidneys
Leucocytes
Mammary
tissue 1
Mammary
tissue 2
Bladder
tissue
Colonic
tissue
Cervical
tissue
Liver 1
Liver 2
Liver 3
Average
(AVG) 5
reference
genes
3.09
3.45
Original tr.
(OT)
Difference
OT-AVG
New tr.
(NT)
Difference
NT-AVG
Difference
OT-NT
2.16
3.83
-0.93
0.38
2.29
3.82
-0.80
0.37
-0.13
0.01
3.57
3.96
3.44
3.46
3.31
3.54
3.45
3.61
3.68
4.18
-0.03
-0.51
0.17
0.22
0.87
3.38
3.45
3.58
3.71
4.15
-0.19
-0.51
0.14
0.25
0.84
0.16
0.00
0.03
-0.03
0.03
3.81
4.44
0.63
4.47
0.66
-0.03
3.41
3.08
-0.33
2.96
-0.45
0.12
2.45
2.04
-0.41
1.78
-0.67
0.26
3.87
3.96
0.09
3.99
0.12
-0.03
2.56
2.00
2.31
3.03
2.85
3.21
0.47
0.85
0.90
3.08
2.76
3.13
0.52
0.76
0.82
-0.05
0.09
0.08
Table 1.6
22
Transcript detection of the AHR gene in the dog
Length new transcript
After gel electrophoresis sample 5, 8, 9 and 11 showed a band for the break4 forward, 10 reverse
primerset (Figure 23) These samples were used to perform sequencing analysis. The found
sequences consisted of exon 7, 8, 9 and 10. For the exact sequences I refer to attachment 2.6.
100 bp
4
5
8
9
10
11
Fig. 23. 4 samples showed one band
for the break forward, 10 reverse primerset.
Confirmation new transcript
After gel electrophoresis all samples showed a small band for each of the three primersets. All PCR
products visible are around 250 basepairs long (Figure 24 and 25) The found sequences consisted
of exon 2, 7, 8 and 9 and are similar to the sequences found using the break4 forward, 9 reverse
primerset. For the exact sequences I refer to attachment 2.7. No differences were found between
the three primersets.
100 bp 5
8
9
11
12
13
100 bp 5
8
9
11
12
14
Fig. 25. Unfortunately, only faint bands were
visible after PCR and gel electrophoresis
using the break3 forward, 9 reverse
primerset.
100 bp 5
8
9
11
12
13
Fig. 24. All samples showed a band of
approximately 250 bp after performing PCR
and gel electrophoresis using the break1 forward,
9 reverse (above) and break2 forward, 9 reverse
primerset (below)
23
Transcript detection of the AHR gene in the dog
Western blotting
After our primary anti-AHR antibody and our secondary anti-rabbit antibody had bound many nonspecific bands were visible (Figure 26) Our original transcript should be visible around 96kD, but
unfortunately no clear band is visible. The same goes for the new transcript, which (if translated)
should have been visible around 12kD. Tubulin on the other hand, which has a molecular weight
around 50-55kD, could be visualized more easily, but the non-specific bands remained visible
(Figure 27) In both figures, fully to the left is our ladder, then 6 samples from healthy beagles,
next a ladder, 6 samples from Irish Wolfhounds, 2 from Cairn terriers, 1 from a Golden retriever
and fully to the right a ladder.
150
100
75
50
37
25
20
15
Fig. 26. Unfortunately, many non-specific bands
were visible, instead of one (and possibly two)
bands as we expected.
150
100
75
50
37
25
20
15
Fig. 27. A clear band around 55kD is visible, apart
from al the non specific bands, which is probably
our control tubulin.
24
Transcript detection of the AHR gene in the dog
Discussion
The existence of another transcript has definitely been proven with both our qPCR data and our
sequencing results using the break1 forward, break2 forward, break3 forward combined with the 9
reverse primer. As for most tissues, except for liver and mammary tissue, just one cDNA sample
was used for performing qPCR no conclusions can be drawn about the relative level of expression
of this new transcript compared to the original transcript. But, as differences in the level of
expression between the new and original transcript were measured, especially in pancreas, uterus,
bladder and colon, this supports the existence of another transcript besides the original transcript
of the AHR gene in the dog. An important note to include about our qPCR data is the fact that in
each tissue sample both the original and new transcript consistently show a higher or lower level
of expression compared to the relative level of expression of our reference genes. So more
research needs be done using more samples per tissue to perform qPCR. This way, a conclusion
could also be drawn about the relative level of expression of the new transcript compared to the
original transcript in different tissues. By looking at the intensity of the bands in Figure 13, where
the PCR products of the 1 forward, 9 reverse primerset are made visible, it is safe to say that the
original transcript is expressed in higher levels than the new transcript in liver tissue, as these
bands are far more intense.
Interestingly, both the qPCR data and visible bands on gel after performing a PCR using cDNA
from mammary tissue, colonic tissue, bladder tissue and cervical tissue support the presence of
the new transcript in these tissues, which differs from Genecards’ results (Figure 1) As no AHR
expression in retinal tissue was measured by Genecards, it would be interesting to perform PCR
and qPCR using cDNA from retinal tissue as well.
As exon 2, 7, 8, 9 and 10 were found in our sequencing results for the new transcript, it would be
easy to say that the new transcript consists of exon 1 (same start codon as the original
transcript), 2, 7, 8, 9 and 10. But, a measurement of the number of basepairs which is left out
between exon 2 and 7 proved that 713 basepairs are left out, which means that a frameshift takes
place. Therefore another stop codon takes effect, which referring to our data should be located in
exon 7. This could mean that the new transcript undergoes nonsense mediated decay. So the new
transcript could consist of exon 8, 9 and 10 also, but as a premature stop codon is present in exon
7, the mRNA will be degraded. This also explains why the bands of the original transcript were far
more prominent than those of the new transcript. As its mRNA is degraded the new transcript will
be found in lower concentrations. On the other hand, our qPCR data did not show big differences
in the relative level of expression between the original and new transcript in liver tissue. To really
prove this new transcript undergoes nonsense mediated decay further research needs to be done
using a cell-line which expresses AHR and suppressing the nonsense mediated decay process.
The results of our Western blotting showed no band for the new transcript, this could mean that
indeed the small transcript is degraded before translated, or the concentration of this protein was
just too low and could thus not be visualized. Another possibility is that the conditions were not
optimal, as 12kD is below in the blot. Also, many non-specific bands were visible, so it is hard to
distinguish these non-specific bands from our specific AHR and tubulin bands. A way to distinguish
these bands is to incubate a piece from the gel with only the secondary antibody. All that would be
stained would be non-specific. Also, it would be better if two different gels were used for the two
transcripts as the molecular weight between the two transcripts differs 84kD, so the original
transcript was at the top of the blot, while the new transcript was below in the blot, both not an
optimal condition.
Referring to sequencing the original transcript, the results would have been even better if overlap
was found between the forward and reverse primer. Now, only the 6 forward and 10 forward
primers gave good results, with the CAG-repeated ‘intron’ found in our cDNA, while the 11 reverse
primer failed.
Last, to get a more complete picture about the conservation of the AHR gene on genetic level,
more species could be compared. Again, nucleotide sequence and aminoacid sequence could be
compared.
25
Transcript detection of the AHR gene in the dog
Conclusion
The first hypothesis, that the AHR gene is highly conserved, would be fairly true if only the results
of comparing the different species’ nucleotide sequences would be taken into account. The lowest
query coverage found was 67% while the highest was 90%. As 67% is considered low, but 90%
high, AHR would be a fairly conserved gene. The results of comparing the different species’
aminoacid sequence lead to a different conclusion as query coverages ranging from 47% to 91%
were found. As 47% is very low the first hypothesis should be rejected. As the aminoacid
sequence ultimately determines the shape of the protein these results count heavier than those of
comparing the different species’ nucleotide sequence.
Interesting is the fact that mouse and rat are much alike (84%-91%, taking both nucleotide
sequence and aminoacid sequence into account) and human and dog (84%-87%), while the three
transcripts of zebrafish were not very comparable to that of any species.
The second hypothesis, namely that there are multiple transcripts of the AHR gene in the dog has
definitely been proven. Our sequencing results and qPCR data are convincing enough to confirm
this hypothesis. Not only did we prove the existence of another transcript besides the original
transcript by performing PCR using the break1 forward, break2 forward, break3 forward and
break4 forward, 9 reverse primersets and performing qPCR, we also detected the presence of this
new transcript in the following tissues: pancreas, adrenal gland, uterus, ovary, kidneys,
leucocytes, mammary tissue, bladder tissue, colonic tissue, cervical tissue and liver tissue. The
level of expression of the new transcript compared to the original transcript still has to be
measured.
Based on our sequencing results, the new transcript consists of exon 1, 2, 7, 8, 9 and 10, but with
a premature stop codon in exon 7. This has led to the idea that this transcript undergoes nonsense
mediated decay and its mRNA is thus degraded in the cell. The results of the Western blotting
support this hypothesis as the new transcript could not be visualized. But, as these results were
not very convincing further research needs to be done to prove this hypothesis.
Last, our sequencing results for the original transcript proved that the intron between exon 10 and
11 is not an intron. So exon 11 in the dog is part of exon 10. This means that, just like the human
transcript, the original transcript in the dog consists of 11 exons.
26
Transcript detection of the AHR gene in the dog
References
[1] Lahvis G.P, Lindell S.L, Thomas R.S, McCuskey R.S, Murphy C, Glover E, Bentz M, Southard J,
Bradfield C.A. Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon
receptor-deficient mice. Proc. Natl. Acad. Sci. U S A. 2000 Sep. 12;97(19):10442-7.
[2] Zhang N. The role of endogenous aryl hydrocarbon receptor signaling in cardiovascular
physiology. J. Cardiovasc. Dis. Res. 2011 Apr;2(2):91-5.
[3] Dietrich C, Kaina B. The aryl hydrocarbon receptor (AhR) in the regulation of cell-cell contact
and tumor growth. Carcinogenesis. 2010 Aug;31(8):1319-28
[4] http://www.genecards.org/cgi-bin/carddisp.pl?gene=AHR&search=ahr
[5] Dolwick K.M, Schmidt J.V, Carver L.A, Swanson H.I, Bradfield C.A. Cloning and expression of a
human Ah receptor cDNA. J. Cardiovasc Dis. Res. 2011 Apr;2(2):91-5.
[6] Sánchez-Martín F.J, Fernández-Salguero P.M, Merino J.M. Novel cDNA sequences of aryl
hydrocarbon receptors and gene expression in turtles (Chrysemys picta and Pseudemys scripta)
exposed to different environments. J. Neurochem. 2011 Jul;118(1):153-62.
[7] Falahatpisheh M.H, Nanez A, Ramos K.S. AHR Regulates WT1 Genetic Programming During
Murine Nephrogenesis. Mol. Med. 2011 Aug. 18.
[8] Kadow S, Jux B, Zahner S.P, Wingerath B, Chmill S, Clausen B.E, Hengstler J, Esser C. Aryl
hydrocarbon receptor is critical for homeostasis of invariant gammadelta T cells in the murine
epidermis. J. Immunol. 2011 Sep 15;187(6):3104-10.
[9] Hooper L.V. You AhR What You Eat: Linking Diet and Immunity. Cell. 2011 Oct.
28;147(3):489-91.
[10] Mimura J, Fujii-Kuriyama Y. Functional role of AhR in the expression of toxic effects by TCDD.
Biochim. Biophys. Acta. 2003 Feb. 17;1619(3):263-8.
[11] Andersson P, et al. A constitutively active dioxin/aryl hydrocarbon receptor induces stomach
tumors. Proc. Natl Acad. Sci. USA 2002;99:9990-9995.
[12] Eagle H, et al. Growth regulatory effects of cellular interaction. Nature 1967;213:1102-1106.
[13] Yang X, et al. The aryl hydrocarbon receptor constitutively represses c-myc transcription in
human mammary tumor cells. Oncogene 2005;24:7869-7881.
[14] Villano CM, et al. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces matrix
metalloproteinase (MMP) expression and invasion in 2058 melanoma cells. Toxicol. Appl.
Pharmacol. 2006;210:212-224.
[15] Chang JT, et al. Requirement of aryl hydrocarbon receptor overexpression for Cyp1B1 upregulation and cell growth in human lung adenocarcinomas. Clin. Cancer Res. 2007;13:38-45.
[16] Ishida M, et al. Activation of the aryl hydrocarbon receptor pathway enhances cancer cell
invasion by up-regulating the MMP expression and is associated with poor prognosis in upper
urinary tract urothelial cancer. Carcinogenesis 2010;31:287-296.
[17] Dietrich C, et al. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-dependent release from contact
inhibition in WB-F344 cells: involvement of cyclin A. Toxicol. Appl. Pharmacol. 2002a;183:117126.
[18] Münzel P, et al. Growth modulation of hepatocytes and rat liver epithelial cells (WB-F344) by
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Carcinogenesis 1996;17:197-202.
27
Transcript detection of the AHR gene in the dog
[19] Chramostova K, et al. Polycyclic aromatic hydrocarbons modulate cell proliferation in rat
hepatic epithelial stem-like WB-F344 cells. Toxicol. Appl. Pharmacol. 2004;196:136-148.
[20] Vondrácek J, et al. Induction of aryl hydrocarbon receptor-mediated and estrogen receptormediated activities, and modulation of proliferation by dinaphthofurans. Environ. Toxicol. Chem.
2004;23:2214-2220.
[21] Vondrácek J, et al. Aryl hydrocarbon receptor-activating polychlorinated biphenyls and their
hydroxylated metabolites induce cell proliferation in contact-inhibited rat liver epithelial cells.
Toxicol. Sci. 2005;83:53-63.
[22] Zatloukalová J, et al. β-Naphthoflavone and 3’-methoxy-4’-nitroflavone exert ambiguous
effects on Ah receptor-dependent cell proliferation and gene expression in rat liver, stem-like’
cells. Biochem. Pharmacol 2007;73:1622-1634.
[23] Weiss C, et al. TCDD deregulates contact inhibition in rat liver oval cells via Ah receptor, JunD
and cyclin A. Oncogene 2008;27:2198-2207.
[24] Diry M, et al. Activation of the dioxin/aryl hydrocarbon receptor (AhR) modulates cell
plasticity through a JNK-dependent mechanism. Oncogene 2006;25:5570-5574.
[25] http://www.ensembl.org/index.html
[26] http://www.ncbi.nlm.nih.gov/
[27] RNeasy® Mini handbook QIAGEN©, 2001-2010.
[28] Genorm
28
Transcript detection of the AHR gene in the dog
Attachment 1.1 RNA isolation protocol
Purification of Total RNA from Animal Tissues
Things to do before starting
β-Mercaptoethanol (β-ME) must be added to Buffer RLT before use. Add 10 μl
β-ME per 1 ml Buffer RLT. Dispense in a fume hood and wear appropriate
protective clothing. Buffer RLT containing β-ME can be stored at room temperature
for up to 1 month.
Alternatively, add 20 μl of 2 M dithiothreitol (DTT) per 1 ml Buffer RLT. The stock
solution of 2 M DTT in water should be prepared fresh or frozen in single-use
aliquots. Buffer RLT containing DTT can be stored at room temperature for up to
1 month.
Buffer RPE is supplied as a concentrate. Before using for the first time, add
4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a working
solution.
If performing optional on-column DNase digestion, prepare DNase I stock solution.
Procedure
1. Excise the tissue sample from the animal or remove it from storage. Remove
RNAlater stabilized tissues from the reagent using forceps. Determine the amount of tissue. Do
not use more than 30 mg.
2. For unstabilized fresh or frozen tissues:
If using the entire tissue, place it directly into a suitably sized vessel for disruption
and homogenization, and proceed immediately to step 3.
If using only a portion of the tissue, weigh the piece to be used, and place it into
a suitably sized vessel for disruption and homogenization. Proceed immediately to step 3.
3. Disrupt the tissue and homogenize the lysate in Buffer RLT (do not use more than 30 mg
tissue) according to step 3a, 3b, 3c, or 3d.
Disruption using a mortar and pestle followed by homogenization using a
QIAshredder homogenizer:
Immediately place the weighed frozen tissue in
liquid nitrogen, and grind thoroughly with a mortar and pestle. Decant tissue
powder and liquid nitrogen into an RNase-free, liquid-nitrogen–cooled, 2 ml
microcentrifuge tube (not supplied). Allow the liquid nitrogen to evaporate, but do
not allow the tissue to thaw.
Add the appropriate volume of Buffer RLT (see Table 1.4). Pipet the lysate directly
into a QIAshredder spin column placed in a 2 ml collection tube, and centrifuge for
2 min at full speed. Proceed to step 4.
Amount of starting material (mg)
<20
20-30
Volume of buffer RLT (µl)
350 or 600
600
Table 1.1.1 Volumes of Buffer RLT for Tissue Disruption and Homogenization
4. Centrifuge the lysate for 3 min at full speed. Carefully remove the supernatant by
pipetting, and transfer it to a new microcentrifuge tube (not supplied). Use only thissupernatant
(lysate) in subsequent steps.
5. Add 1 volume of 70% ethanol* to the cleared lysate, and mix immediately by
pipetting. Do not centrifuge. Proceed immediately to step 6.
29
Transcript detection of the AHR gene in the dog
* Using 50% ethanol (instead of 70% ethanol) may increase RNA yields from liver samples.
6. Transfer up to 700 μl of the sample, including any precipitate that may have
formed, to an RNeasy spin column placed in a 2 ml collection tube. Close
the lid gently, and centrifuge for 15s at ≥8000 x g (≥10,000 rpm). Discard the
flow-through.
Reuse the collection tube in step 7.
If the sample volume exceeds 700 μl, centrifuge successive aliquots in the same
RNeasy spin column. Discard the flow-through after each centrifugation.†
Optional: If performing optional on-column DNase digestion (Eliminating
genomic DNA contamination), follow steps D1–D4 after
performing this step.
Optional On-column DNase Digestion with the RNase-free DNase Set
Things to do before starting
Prepare DNase I stock solution before using the RNase-Free DNase Set for the first
time. Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 μl of the RNasefree
water provided. To avoid loss of DNase I, do not open the vial. Inject RNasefree
water into the vial using an RNase-free needle and syringe. Mix gently by
inverting the vial. Do not vortex.
D1. Add 350 μl Buffer RW1 to the RNeasy spin column. Close the lid gently, and
centrifuge for 15s at ≥8000 x g (≥10,000 rpm) to wash the spin column
membrane. Discard the flow-through.
D2. Add 10 μl DNase I stock solution (see above) to 70 μl Buffer RDD. Mix by gently
inverting the tube, and centrifuge briefly to collect residual liquid from the sides of
the tube.
D3. Add the DNase I incubation mix (80 μl) directly to the RNeasy spin column
membrane, and place on the benchtop (20–30°C) for 15 min.
D4. Add 350 μl Buffer RW1 to the RNeasy spin column. Close the lid gently, and
centrifuge for 15s at _8000 x g (_10,000 rpm). Discard the flow-through.
Continue with the first Buffer RPE wash step in the relevant protocol.
7. Add 700 μl Buffer RW1 to the RNeasy spin column. Close the lid gently, and
centrifuge for 15s at ≥8000 x g (≥10,000 rpm) to wash the spin column
membrane. Discard the flow-through.
Skip this step if performing optional on-column DNase digestion.
8. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and
centrifuge for 15s at ≥8000 x g (≥10,000 rpm) to wash the spin column
membrane. Discard the flow-through.
9. Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and
centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin column
membrane.
10. Optional: Place the RNeasy spin column in a new 2 ml collection tube (supplied),
and discard the old collection tube with the flow-through. Close the lid gently, and
centrifuge at full speed for 1 min.
11. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add
30–50 μl RNase-free water directly to the spin column membrane. Close the lid
gently, and centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the RNA.
12. If the expected RNA yield is >30 μg, repeat step 11 using another 30–50 μl RNasefree
water, or using the eluate from step 11 (if high RNA concentration is required).
Reuse the collection tube from step 11. [27]
30
Transcript detection of the AHR gene in the dog
Attachment 1.2 Bio-Rad cDNA synthesis protocol
This protocol requires the Bio-Rad iScript cDNA Synthesis Kit.
Reaction set up:
Component
Volume per reaction
5x iScript Reaction Mix
iScript Reverse Transcriptase
Nuclease-free water
RNA template (100fg to 1μg Total RNA)*
4μl
1μl
xμl
xμl
Total Volume
20 μl
Incubate complete reaction mix:
Time (min)
5
30
5
∞
Temperature (°C)
25
42
85
4
31
5μl
15μl in total
10μl=0.5μg RNA
Transcript detection of the AHR gene in the dog
Attachment 1.3 PCR amplification protocol
25µl reaction
Reaction set up:
Component
10x PCR Buffer
MgCl2 (50 mM)
dNTP’s (1 mM)
Primer F (10 µM)
Primer R (10µM)
Platinum Taq
mQ
cDNA
Volume per
reaction (µl)
2.5
1
5
1.25
1.25
0.25
12.75
1
Concentration
1x
2 mM
200 µM
0.5 µM
0.5 µM
***
***
***
x Mix
2.5x
x
5x
1.25x
1.25x
0.25x
12.75x
***
Incubate complete reaction mix:
Temperature (°C)
95
95
50-65*
72
72
20
Time
5 min
30s
30s
15s-1.5 min#
10 min
∞
* depends on the primerset
# depends on polymerase and size cDNA
32
35-45 cycles
Transcript detection of the AHR gene in the dog
Attachment 1.4 SAP/ExoI purification protocol
14µl reaction
Reaction set up:
Component
µl SAP (1U/µl)
µl ExoI (20U/µl)
mQ
µl PCR product
Total
Volume per reaction (µl)
3.25
0.075
0.675
10
14
Incubate complete reaction mix:
Time (min)
60
20
∞
Temperature (°C)
37
75
12
33
x Mix
3.25x
0.075x
0.675x
***
***
Transcript detection of the AHR gene in the dog
Attachment 1.5 Big Dye Terminator Cycle Sequencing Protocol
Reaction set up:
Component
Terminator Ready Reaction
Mix (BDT)
Template: PCR product
(purified or diluted)
Plasmid DNA: 100-200 bp
200-500 bp
500-1000 bp
1000-2000 bp
Primer, 3.2 pmol
5x Sequence Buffer
MilliQ
Total Volume
Volume per reaction (µl)
1
x Mix
X
2 µl
2x
1-3 ng
3-10 ng
5-20 ng
10-40 ng
1 µl
2 µl
4 µl
10 µl
X
2x
4x
10x
Step 2 Mix well and spin briefly.
Cycle sequencing on the Gene Amp PCR-System 9700 or MJ Research Dyad Disciple Peltier
Thermal Cycler
Step
1
2
3
4
5
Action
Place the tubes in a thermal cycler: with
heated lid.
Repeat the following for 35 cycles:
Rapid thermal ramp to 96°C
96°C for 30s
Rapid thermal ramp to 50°C
50°C for 15s (depends on the
annealing temperature in PCR)
Rapid thermal ramp to 60°C
60°C for 1.5 min
Rapid thermal ramp to 4°C and hold ready
to purify
Spin down the contents of the tubes in a
microcentrifuge
Proceed to Sephadex purification
Sephadex purification:
Dye Terminator Removal Using Multiscreen 96-Well Filtration Plates
Step
1
2
Action
Load dry Sephadex into all 96-wells of a
Multiscreen MAHV plate using the column
loader as follows:
Add Sephadex G-50 to the Column
Loader
Remove excess resin off the top of
the column loader with the scraper
Place Multiscreen MAHV plate
upside-down on the top of the
Column Loader
Invert both Multiscreen MAHV plate
and the Column Loader
Tap on top or side of the Column
Loader to release the resin
Using a multi-channel pipettor, add 300 µl
MilliQ water to each well to swell resin.
Incubate at room temperature for 3 hr.
34
Transcript detection of the AHR gene in the dog
3
4
5
6
7
-Once the mini-columns are swollen in
Multiscreen plates, they can be stored in the
refrigerator at 4C for up to two weeks, by
tightly sealing the plates with parafilm
Place a Centrifuge Alignment Frame on top
of a Standard 96-well microplate, then place
the MAHV plate on the assembly, without
lid. Centrifuge at 1900 rpm for 5 min to
pack the mini-columns
Carefully add 10-20 milliQ water to the
sequencing reactions (10 ul, sample), and
pipet everything to the center of the
columns
Tape off the unused mini-columns
Place the MAHV plate (without lid) on top
of a sequencing plate (an MicroAmp Optical
96-Well Reaction Plate) and centrifuge at
1900 rpm for 5 min. (the position of the
samples must correspond with empty wells
in the sequencing plate!!)
Proceed to Electrophoresis on the ABI Prism
3130xl
Electrophoresis on the Prism 3130xl
Step
1
2
Action
Do NOT denaturate the samples
Refer to the Protocol Sequence Sequence
and Genescan; ABI3130XL.
35
Transcript detection of the AHR gene in the dog
Attachment 1.6 Ethanol precipitation protocol
-
Add 10 µl mQ to a tube
Add 5 µl 125 mM EDTA to the (same) tube
Add samples
Add 63 µl 100% EtOH to the EDTA
Mix by turning the tube
Incubate 15 minutes at room temperature
Start working on ice
Spin in centrifuge for 15 minutes at 4ºC to pellet DNA (10.000 rpm)
Aspirate off the fluid using a pipette from glass
Add 60 µl iced 70% EtOH to the precipitate and mix
Spin in centrifuge for 5 minutes at 4ºC (10.000 rpm)
Aspirate off the EtOH using a pipette from glass
Air-dry the DNA in a laboratory bath and resuspend in 20 µl mQ
Use this product for sequencing
36
Transcript detection of the AHR gene in the dog
Attachment 1.7 qPCR protocol
25 µl mQ was added to each sample (4-fold dilution) added to S1.
The following samples were added to S1: pancreas, adrenal gland, uterus, ovary, kidneys,
leucocytes, liver (3 samples)
Standard line:
S1 will contain 7µl of each sample from the 4xcDNA dilution
S1
S2
S3
S4
S5
S6
S7
S8
Volume cDNA (µl)
63
15 S1
15 S2
15 S3
15 S4
15 S5
15 S6
mQ (µl)
45
45
45
45
45
45
60
Primer solutions
New primers are dissolved in mQ to obtain a primary stock solution with a concentration of 100
pmol/µl. The working stocks are 10x dilutions of the primary stock obtaining a 10 pmol/µl
concentration.
Master mix (prepared in the UV cupboard using the SingleChannel pipette, speed 7):
Samples
mQ
iQ SYBR green
Supermix
Forward Primer
Reverse Primer
1x
duplo
26
26
16x duplo
24x duplo
32x duplo
40x duplo
48x duplo
417
417
625
625
833
833
1042
1042
1250
1250
2.35
2.35
38
38
57
57
75
75
95
95
113
113
- After preparing the mastermix 54 µl of the mix is used for each reaction, this should be enough
to ensure a 25µl volume per reaction (duplicates)
- To each 2µl cDNA a volume of 54 µl master mix is added (SingleChannel pipette speed 7)
- Mix by pipetting up and down several times. This can now be pipetted into a BioRad I-cycler
plate in 25 µl duplicate (MultiChannel: PipetteAndMix 25 µl speed 10)
Overview plate:
A
B
C
D
E
F
G
H
1
S1
S2
S3
S4
S5
S6
S7
S8
2
S1
S2
S3
S4
S5
S6
S7
S8
3
X1
X2
X3
X4
X5
X6
X7
X8
4
X1
X2
X3
X4
X5
X6
X7
X8
5
X9
X10
X11
X12
X13
X14
X15
X16
6
X9
X10
X11
X12
X13
X14
X15
X16
7
S1
S2
S3
S4
S5
S6
S7
S8
8
S1
S2
S3
S4
S5
S6
S7
S8
9
X1
X2
X3
X4
X5
X6
X7
X8
10
X1
X2
X3
X4
X5
X6
X7
X8
11
X9
X10
X11
X12
X13
X14
X15
X16
12
X9
X10
X11
X12
X13
X14
X15
X16
- When finished pipetting carefully put on a seal, with clean hands. If using the green 96-well tray
make sure it is not contaminated with SYBRgreen mix, if you suspect it not to be clean rinse with
tap water. Do NOT touch the seal.
- When the plate is sealed, spin briefly. Do NOT touch the top of the plate.
- Place it in the PCR machine, and start your run. Do NOT touch the top of the plate.
37
Transcript detection of the AHR gene in the dog
Attachment 1.8 Western blotting protocol
Gel-electrophoresis
Materials:

0.5M Tris-HCl pH 6.8
Dissolve 12.11 g Tris (B24) in 150 ml mQ
Adjust pH to 6.8 with 6-12M HCl and complement the volume to 200ml

2x sample buffer (8 ml)
2.0 ml 0.5M Tris-HCl pH 6.8
2.0 ml 10% SDS (RT)
1.2 ml 0.05% bromophenolblue ()
2.8 ml glycerol (Sigma G 7757) ()
Add DTT (30 mg/ml) () to the sample buffer just before use.

Electrode buffer (10x concentrated)
144 g glycine (Sigma G 7126) ()
30 g Tris (B24)
100ml 10%SDS
Dissolve in 800ml milliQ, adjust to a pH8.3 and complement the volume to 1L.

Gels: either precast or selfcast.
Procedure:
1. Dilute 50 ml 10x concentrated buffer with 450 ml milliQ just before use (needed for one tank
i.e. 2 gels).
2. Cut the plastic cover from the bottom of the pre-cast gel and take out the comb gently in a
straight upward line.
3. Place the gel cassettes in the electrode assembly
4. Place electrode assembly in clamping frame.
5. Close the two cam levers of the clamping frame.
6. Lower the inner chamber into the mini tank
7. Fill the inner chamber and part of the tank with 1x electrode buffer
8. Important! flush the wells with electrode buffer
9. Dilute the samples to the desired concentration (eg. approx. 2µg/µl). This solution is diluted
with 2x sample buffer (containing DTT) (1:1). The average final concentration is
approximately 1µg/µl.
10. Heat the samples at 95ºC for 2 min.
11. Pipet the samples in the slots. To at least one lane load 10 µl of the Precision plus Protein
Standard (dual color) to follow the electrophoresis. For easy identification of left and right
side of your gel, load the marker asymmetrically. Fill any unused slots with 1x sample buffer.
12. Run the gel on 100 – 150 Volts for as long needed to sufficient separation of your marker
bands. (possible to max 200 Volts (max 0.04A) Lower voltage settings will give a nicer /
straighter front
Blotting
Materials:

Blotbuffer 1 liter (pH= 8.3) Prepare (fresh @ 4°C)
3.03 g Tris (25mM) (B24)
14.4 g glycine (192 mM) (B17)
200 ml methanol (chemical quality, Boom)
Fill up with milliQ to 1 liter, this enough for 1 blotting system i.e. blotting 2 gels
It is not necessary to adjust the pH.
Procedure:
1. Cut the blot membrane to the dimension of the gel
38
Transcript detection of the AHR gene in the dog
2.
3.
4.
When the electrophoresis is finished, decant the electrode buffer in to the mini tank.
Dissemble the electrophoresis assembly
Pour some blot-buffer in 3 glass containers and put the gaskets and the filter paper in one,
the membrane in to another and the gels in to the third one, let them soak for at least 15
minutes, preferably 30 minutes.
To remove the big glass-plate from the gel follow these steps
Cut the plastic on one side on the white print, cut between the two glass plates.
Put the side you just cutted down on the table.
Using the same knife; gently slide it between the two glass plates and twist the
blade so the 2 glass plates come apart.
Slowly lift the big lass plate make sure the gel sticks to the small one
Cut the slots from the top of the gel and the rim at the bottom, make sure you get
rid of all pieces of gel
Prepare the gel sandwich.
Place the cassette with the black side down in a tray filled with some buffer
On the black side place one soaked fiber pad
On the fiber pad place a soaked filter paper
On the filter paper place the gel, remove the air bubbles)*
Place the membrane on the gel, remove the air bubbles)*
Place a soaked filter paper on the membrane, remove air bubbles )*
Complete the sandwich by adding a last soaked fiber pad
)* Remove all air bubbles by gently rolling a glass rod over it; make sure all air is
rolled out as this negatively influences the results.
Close the cassette, lock it closed with white latch
Place the cassette in the module (black to black).
Repeat previous steps (6 till 8) for the other cassette.
Place the module in the tank; add a stir bar, to help maintain even buffer temperature and
ion distribution in the tank.
Add a frozen ice cooling unit and fill the tank completely with cold blotting buffer
Put on the lid, plug the cables into the power supply (black in black)
Blot for 1 hour @ 100V. (check if there is any current)
5.
6.
7.
8.
9.
10.
11.
12.
After protein transfer
1. Remove cooling unit and blotting module from the tank
2. Open the cassettes and disassemble the sandwich
3. Mark orientation of the gel on the protein side of the blot
4. Proceed to the next step; immuno detection or dry your membrane on a piece of saran wrap
placed between a sheet of filtration paper.
5. Clean all components with tap water and rinse with demi-water.
6. Dispose the blotbuffer in the proper waste vessel.
Staining a blot with the ECL Advanced Detection kit
Materials:

10x TBS pH 8.0:
24.22 g Tris (0.2M) (B24)
87.7 g NaCl (1.5M) (B20)
Dissolve in 900 ml water
Adjust pH to 8.0 with HCl.
Fill it up to 1liter with milliQ
Keep it at 4ºC.

TBST-buffer (0.1 % Tween) (TBST0.1%)
100 ml 10x TBS (4°C)
Add the volume to 1liter with milliQ
Add 1 ml Tween 20
Keep it at 4ºC.
39
Transcript detection of the AHR gene in the dog

ECL Advanced Western Blotting Detection Kit (Amersham RPN2135)

TBST + 4% Blocking Solution
Dissolve 4 g ECL Blocking Solution powder in 100 ml TBST.

Working Solution.
Mix detection solutions A and B in a ratio of 1:1, for 2 blot add 2.5 ml solution A to
2.5 ml of solution B.
(Solution stable for 1 month if kept in the dark and at 4°C)
 Note that these concentrations and type of blocking are the ones mostly used. The blocking
concentrations can vary between 1 and 5%. Other blocking proteins can be used, mostly used are
BSA and non-fat dry-milk.
Procedure:
* Perform all the steps at room temperature and under constantly (gently) shaking.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Block the membrane 60 minutes in TBST0.1% + 4% Blocking (ECL). Or other concentration
or blocking-protein-solution.
Overnight incubation at 4°C in TBST0.1% + primary antibody (in 4% BSA) (re-use is
possible).
Wash 3 x 5 minutes in TBST0.1%, if you need stronger washing, to decrease background
staining, increase the percentage of tween in your wash buffer
Incubate 60 minutes in your HRP conjugated secondary antibody dissolved at the desired
concentration in TBST0.1%
Wash 3 x 5 minutes in TBST0.1%
Drain of Wash buffer, place protein side up on piece of copier sheet.
Place blot on sheet on the tray of the chemi-doc
Incubate with the substrate solution
Acquire image for 10 seconds
look for the most intense band, measure it’s density using the density tools
For the time of the next exposure calculate: 4000/(Average density)x 10 seconds
Set this as the exposure time and click manual expose
40
Transcript detection of the AHR gene in the dog
Attachment 2.1 Sequencing results
PCR followed by nested PCR
Sequencing results after the first PCR with primerset 1 forward, 9 reverse
Sample 4, 1 forward primer
ACTGTCAAGCCAATCCCAGCTGAAGnATCAAGTCAnATCCTTCCAAGCGACATAGAGACCnACTTAATACAG
AGTTGGACCGTTTGGCTAGTCTGCTGCCTTTTCCACAAGATGTTATTAATAnGCTGGACAAACTTTCAGTGCT
TAnnCTCAGTGTCAGTTACCTAAGGGCCAAGAGCTTCTTTGATGTTGCATTACAnTCCTCCCCAACTGACAGA
AATGAAGTCCAGGAAAACTGTnGAACAAAATTCAGAGAAGGTCTGCATCTGCnnGAAGGAGAATTCTTATTA
CAGGCTCTGAATGGnTTTGTGCTGGTTGTCACCACnGATGCTTTGGTCTTTTATGCTTCTTCTACCATACAAG
ATTACCTAGGGTTTCAGCAGTCTGATGTCATACATCAGAGCGTATATGAACTTATTCATACTGAAGACCGAG
GTGAATTTCAGCGTCAGCTACACTGGACATTAAACCCTTCACAGTGTACAGACTCTGGACAAGGAGTTGATG
ACGCTAATGGGCTGCCACAGCCAGTAGTCTGTTATAACCCAGACCAGCTTCCTCCAGAAAACTCTTCCTTAA
TGGAAAGGAGCTTCGTGTGCCGACTAAGGTGTCTGCTGGATAATTCGTCCGGTTTTCTGGCAATGAATTTCC
A
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
1
2
3
4
5
6
7
8
Sample 4, 9 reverse primer
CTAAGGTGTCTGCnGGATAATTCGTCCGGTTTTCTGGCAATGAATTTCCAAGGGAGGTTAAnGTATCTTCnT
GGACAGAnCAAGAAAGGGAAAGATGGnTCAATnCTGCCACCTCAGTTGGCTTTGTTTGCAATAGCTACTCCA
CTTCAACCACCATCCATCCTTGnGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTA
CACCTACTGCnTGTGATGCCAAAGGAAAACnTGTTTTAGGCTATAnTGAAGCAGAGTnGTGCATGAGGGGAT
CAGGATACCAATTTATTCATGCTGCTGATATGCTTATGT
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
ACTGTCAAGCCAATCCCAGCTGAAGAATCAAGTCAAATCCTTCCAAGCGACATAGAGACCGACTTAATACAG
AGTTGGACCGTTTGGCTAGTCTGCTGCCTTTTCCACAAGATGTTATTAATAAGCTGGACAAACTTTCAGTGCT
TAGGCTCAGTGTCAGTTACCTAAGGGCCAAGAGCTTCTTTGATGTTGCATTACAGTCCTCCCCAACTGACAG
AAATGAAGTCCAGGAAAACTGTAGAACAAAATTCAGAGAAGGTCTGCATCTGCAAGAAGGAGAATTCTTATT
ACAGGCTCTGAATGGCTTTGTGCTGGTTGTCACCACAGATGCTTTGGTCTTTTATGCTTCTTCTACCATACAA
GATTACCTAGGGTTTCAGCAGTCTGATGTCATACATCAGAGCGTATATGAACTTATTCATACTGAAGACCGA
GGTGAATTTCAGCGTCAGCTACACTGGACATTAAACCCTTCACAGTGTACAGACTCTGGACAAGGAGTTGAT
GACGCTAATGGGCTGCCACAGCCAGTAGTCTGTTATAACCCAGACCAGCTTCCTCCAGAAAACTCTTCCTTA
ATGGAAAGGAGCTTCGTGTGCCGACTAAGGTGTCTGCTGGATAATTCGTCCGGTTTTCTGGCAATGAATTTC
CAAGGGAGGTTAAAGTATCTTCATGGACAGAACAAGAAAGGGAAAGATGGTTCAATACTGCCACCTCAGTT
GGCTTTGTTTGCAATAGCTACTCCACTTCAACCACCATCCATCCTTGAGATCCGAACCAAAAATTTCATCTTT
AGAACCAAACACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTG
AAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTATGT
Sample 8, 1 forward primer
CCAnCTGAAGGAnTCAAGTCAnATCCTTCCAAGCGACATAGAGACCGACTTAATACAGAGTTGGACCGTTTG
GCTAGTCTGCTnnCTTTTCCACAAGATGTTATTAATAAGCTGGACAAACTTTCAGTGCTTAGGCTCAGTGTCA
GTTACCTAAGGGCCAAGAGCTTCTTTGATGTTGCATTACAGTCCTCCCCAACTGACAGAAATGAAGTCCAGG
AAAACTGTnGAACAAAATTCAGAGAAGGTCTGCATCTGCAnGAAGGAnAATTCTTATTACAGGCTCTGAATG
GnTTTGTGCTGGTTGTCACCACAGATGCTTTGGTCTTTTATGCTTCTTCTACCATACAAGATTACCTAGGGTT
TCAGCAGTCTGATGTCATACATCAGAGCGTATATGAACTTATTCATACTGAAGACCGAGGTGAATTTCAGCG
41
Transcript detection of the AHR gene in the dog
TCAGCTACACTGGACATTAAACCCTTCACAGTGTACAGACTCTGGACAAGGAGTTGATGACGCTAATGGGCT
GCCACAGCCAGTAGTCTGTTATAACCCAGACCAGCTTCCTCCAGAAAACTCTTCCTTAATGGAAAGGAGCTT
CGTGTGCCGACTAAGGTGTCTGCTGGATAAT
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
1
2
3
4
5
6
7
8
Sample 8, 9 reverse primer
GGATAATTnGTCCGGTTnTnTnGCAATGAATTTnCAAGGGAGGTTnAAGTATCTTCATGGACAGAnCnAGAAA
GGGAAAGATGGTTCAATnnTGCCACCTCAGTTGGCTTTGTTTGCAATAGCTACTCCACTTCAACCACCATCCA
nCCTTGAGATCCGAACCnAAAATTTCATCTTTAGAACCAAACnCAAACTAGACTTTACACCTACTGCnTGTGA
TGnnAAAGGAAAACnTGTTTTAGGCTATAnTGAAnCnGAGTTGTGCATGAGGGGAnCAGGATnCCAATTTATT
CATGCTGCTGATATG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
CCAGCTGAAGGAATCAAGTCAAATCCTTCCAAGCGACATAGAGACCGACTTAATACAGAGTTGGACCGTTTG
GCTAGTCTGCTGCCTTTTCCACAAGATGTTATTAATAAGCTGGACAAACTTTCAGTGCTTAGGCTCAGTGTCA
GTTACCTAAGGGCCAAGAGCTTCTTTGATGTTGCATTACAGTCCTCCCCAACTGACAGAAATGAAGTCCAGG
AAAACTGTAGAACAAAATTCAGAGAAGGTCTGCATCTGCAAGAAGGAGAATTCTTATTACAGGCTCTGAATG
GCTTTGTGCTGGTTGTCACCACAGATGCTTTGGTCTTTTATGCTTCTTCTACCATACAAGATTACCTAGGGTT
TCAGCAGTCTGATGTCATACATCAGAGCGTATATGAACTTATTCATACTGAAGACCGAGGTGAATTTCAGCG
TCAGCTACACTGGACATTAAACCCTTCACAGTGTACAGACTCTGGACAAGGAGTTGATGACGCTAATGGGCT
GCCACAGCCAGTAGTCTGTTATAACCCAGACCAGCTTCCTCCAGAAAACTCTTCCTTAATGGAAAGGAGCTT
CGTGTGCCGACTAAGGTGTCTGCTGGATAATTnGTCCGGTTTTCTGGCAATGAATTTCCAAGGGAGGTTAAA
GTATCTTCATGGACAGAACAAGAAAGGGAAAGATGGTTCAATACTGCCACCTCAGTTGGCTTTGTTTGCAAT
AGCTACTCCACTTCAACCACCATCCATCCTTGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAA
CTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGC
ATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATG
Sample 11, 1 forward primer
ATCCCAGCTGAGGAnCAAGTCnATCCTTCCAAGCGACATAGAnnCnnACTTAATACAGAGTTGGACCGTTTG
GCTAGTCTnCnGCCTTTTCCACAAGATGTTATTAATAAGCTGGACAAACTTTCAGTGCTTAGGCTCAGTGTCA
GTTACCTAAGGGCCAAGAGCTTCTTTGATGTTGCATTACAGTCCTCCCCAACTGACAGAAATGAAGTCCAGG
AAAACTGTAGAACAAAATTCAGAGAAGGTCTGCATCTGCAAGAAGGAGAATTCTTATTACAGGCTCTGAATG
GCTTTGTGCTGGTTGTCACCACAGATGCTTTGGTCTTTTATGCTTCTTCTACCATACAAGATTACCTAGGGTT
TCAGCAGTCTGATGTCATACATCAGAGCGTATATGAACTTATTCATACTGAAGACCGAGGTGAATTTCAGCG
TCAGCTACACTGGACATTAAACCCTTCACAGTGTACAGACTCTGGACAAGGAGTTGATGACGCTAATGGGCT
GCCACAGCCAGTAGTCTGTTATAACCCAGACCAGCTTCCTCCAGAAAACTCTTCCTTAATGGAAAGGAGCTT
CGTGTGCCGACTAAGGTGTCTGCTGGATAATTCGTCCGGTTTTCTGGCAATGAATTTCCAAGGG
Exon
Exon
Exon
Exon
Exon
Exon
Exon
1
2
3
4
5
6
7
Sample 11, 9 reverse primer
TCAGTTACCTAAGGTCAAGAAGCTTCTTTGATGTTGCATTACAGTCCTCCCCAACTGACAGAAATGAAGTCCA
GGAAAACTGTAGAACAAAATTCAGAGAAGGTCTGCATCTGCAAGAAGGAGAATTCTTATTACAGGCTCTGAA
42
Transcript detection of the AHR gene in the dog
TGGCTTTGTGCTGGTTGTCACCACAGATGCTTTGGTCTTTTATGCTTCTTCTACCATACAAGATTACCTAGGG
TTTCAGCAGTCTGATGTCATACATCAGAGCGTATATGAACTTATTCATACTGAAGACCGAGGTGAATTTCAGC
GTCAGCTACACTGGACATTAAACCCTTCACAGTGTACAGACTCTGGACAAGGAGTTGATGACGCTAATGGG
CT
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
ATCCCAGCTGAAGGAATCAAGTCAAATCCTTCCAAGCGACATAGAGACCGACTTAATACAGAGTTGGACCGT
TTGGCTAGTCTGCTGCCTTTTCCACAAGATGTTATTAATAAGCTGGACAAACTTTCAGTGCTTAGGCTCAGTG
TCAGTTACCTAAGGGCCAAGAGCTTCTTTGATGTTGCATTACAGTCCTCCCCAACTGACAGAAATGAAGTCC
AGGAAAACTGTAGAACAAAATTCAGAGAAGGTCTGCATCTGCAAGAAGGAGAATTCTTATTACAGGCTCTGA
ATGGCTTTGTGCTGGTTGTCACCACAGATGCTTTGGTCTTTTATGCTTCTTCTACCATACAAGATTACCTAGG
GTTTCAGCAGTCTGATGTCATACATCAGAGCGTATATGAACTTATTCATACTGAAGACCGAGGTGAATTTCA
GCGTCAGCTACACTGGACATTAAACCCTTCACAGTGTACAGACTCTGGACAAGGAGTTGATGACGCTAATG
GGCTGCCACAGCCAGTAGTCTGTTATAACCCAGACCAGCTTCCTCCAGAAAACTCTTCCTTAATGGAAAGGA
GCTTCGTGTGCCGACTAAGGTGTCTGCTGGATAATTCGTCCGGTTTTCTGGCAATGAATTTCCAAGGG
Sequencing results after the nested PCR with primerset 2 forward, 5 reverse
Sample 8, primerset 2 forward, 5 reverse
CGACTTAATACAGAGTTGGACCGTTTGGCTAGTCTGCTGCCTTTTCCACAAGATGTTATTAATAAGCTGGAC
AAACTTTCAGTGCTTAGGCTCAGTGTCAGTTACCTAAGGGCCAAGAGCTTCTTTGATGTTGATTACAGTCCTC
CCCAnTGACAGAAATGAnnnCCnnnAAAACTGTAnAACAAAATTCAnAGAAGGTCTGCATCTGCAAGAAGGA
GAATTCTTATnACAGGCTCnGAATGGnTTTGTGCTGGTTGTCnCCACAnATGnTTTGGTCTTTTATGCTTCTTC
TACCATACAAGATTACCTAGGGTTnCA
Exon 2
Exon 3
Exon 4
After correcting the n’s:
CGACTTAATACAGAGTTGGACCGTTTGGCTAGTCTGCTGCCTTTTCCACAAGATGTTATTAATAAGCTGGAC
AAACTTTCAGTGCTTAGGCTCAGTGTCAGTTACCTAAGGGCCAAGAGCTTCTTTGATGTTGATTACAGTCCTC
CCCAATGACAGAAATGAAGTCCAGGAAAACTGTAGAACAAAATTCAGAGAAGGTCTGCATCTGCAAGAAGG
AGAATTCTTATTACAGGCTCTGAATGGCTTTGTGCTGGTTGTCACCACAGATGCTTTGGTCTTTTATGCTTCT
TCTACCATACAAGATTACCTAGGGTTTCA
43
Transcript detection of the AHR gene in the dog
Attachment 2.2 Sequencing results
2 forward, 9 reverse
Sample 4, 2 forward primer
ATCCGACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGTGATnCCAAAGG
AAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGC
TGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 4, 9 reverse primer
CCAGCTGAAGGAATCAAGTCAAATCCTTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAAC
CAAACACAAACTAGACTTTACACCTACTGCTTGTGATnCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCA
GAGTnGnGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATAT
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
CCAGCTGAAGGAATCAAGTCAAATCCTTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAAC
CAAACACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCA
GAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCAT
ATCCGGATGATTAAGACAGGAGAGAGTGGCATGA
Sample 5, 2 forward primer
CGACTTAATACAGAGTTGGACCGTTTGGCTAGTCTGCTGCCTTTTCCACAAGATnTTATTAATAAGCTAGACT
TTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGG
GATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGAC
AGGAGAGAGTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The G belongs to exon 2, while the A belongs to exon 7
Sample 5, 9 reverse primer
CCAGCTGAAGGAATCAAGTCAAATCCTTCCAAGCGACATAGAGACCGACTTAATACAGAGTTGGACCGTTTG
GCTAGTCTGCTGCCTTTTCCACAAGATGTTATTAATAAGCTAGACTTTACACCTACTGCTTGTGATGCCAAAG
GAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTG
CTGATATGCTTATTGTGC
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
CGACTTAATACAGAGTTGGACCGTTTGGCTAGTCTGCTGCCTTTTCCACAAGATGTTATTAATAAGCTAGACT
TTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGG
GATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGAC
AGGAGAGAGTGGCATGA
Sample 8, 9 reverse primer
44
Transcript detection of the AHR gene in the dog
GCATATCAGCAGCATGAATAAATTGGTATCCTGATCCCCTCATGCACAACTCTGCTTCAGTATAGCCTAAAAC
AAGTTTTCCTTTGGCATCACAAGCAGTAGGTGTAAAGTCTAGCTTATTAATAACATCTTGTGGAAAAGGCAG
CAGACTAGCCAAACGGTCCAACTCTGTATTAAGTCGGTCTCTATGTCGCTTGGAAGGATTTGACTTGATTCC
TTCAGCTGG
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
The C belongs to exon 2, while the T belongs to exon 7.
Sample 9, 2 forward primer
CCACCATCCATCCTTGnGATCCGAACCAAAAATTTCATCTTTAGAACCAAACAnAnACTAnACTTTACACCTAC
TGCTTGTGATGCCnAAGGAAAACTTGTTTTAGGCTATACTGAAGCAnAGTTGTGCATGAGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAG
TGGCATGAAT
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
Sample 9, 9 reverse primer
TCCAGCTGAAGGAATCAAGTCAAATCCTTCCAAGCGACATAGAGACCGACTTAATACAGAGTTGGCTTTGTT
TGCAATAGnTACTCCACTTCAACCnCCATCCnTCCTTGAGATCCGAACCAAAAATTTCATCnTTAGAACCAAA
CACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAG
TTGTGCATGAGGGGATCAGGATACC
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TCCAGCTGAAGGAATCAAGTCAAATCCTTCCAAGCGACATAGAGACCGACTTAATACAGAGTTGGCTTTGTT
TGCAATAGCTACTCCACTTCAACCACCATCCATCCTTGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAA
CACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAG
TTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCC
GGATGATTAAGACAGGAGAGAGTGGCATGAAT
45
Transcript detection of the AHR gene in the dog
Attachment 2.3 Sequencing results
Break primers
Sample 4, break4forward primer
ACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTT
GTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCG
GATGATTAAGACAGGAGAGAGTGGCATGAATAAATTGG
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 4, 9 reverse primer
GCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTG
TGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATT
TATTCATGCTGCTGATATGCTTTATTGTGC
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
GCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTG
TGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATT
TATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCAT
GAATAAATTGG
Sample 5, break4 forward primer
CAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTT
GTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCG
GATGATTAAGACAGGAGAGAGTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 5, 9 reverse primer
CGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGT
GATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTT
ATTCATGCTGCTGATATGCTTTATTGGT
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
CGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGT
GATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTT
ATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCATG
A
Sample 8, break4 forward primer
TCATGCCACTCTCTCCTGTCTTAATCATCCGGATATGGTACTCAGCACAATAAAGCATATCAGCAGCATGAAT
AAATTGGTATCCTGATCCCCTCATGCACAACTCTGCTTCAnnATAGCCTAAAACAAGTTTTCCTTTGGCATCA
CAAGCAGTAGGTGTAAAGTCTAGTTTGAGTT
46
Transcript detection of the AHR gene in the dog
Sample 8, 9 reverse primer
AAGCATATCAGCAGCATGAATAAATTGGTATCCTGATCCCCTCATGCACAACTCTGCTTCAGTATAGCCTAAA
ACAAGTTTTCCTTTGGCATCACAAGCAGTAGGTGTAAAGTCTAGTTTGAGTTTGGTTCTAAAGATGAAATTTT
TGGTTCGGATCTCTATGTCG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript (reverse complement sequence):
CGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGT
GATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTT
ATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCATG
A
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 9, break4 forward primer
TCATGCCACTnTCTnCTGTCTTAATCATCCGGATATGGTACTCAGCACAATAAAGCATATCAGCAGCATGAAT
AAATTGGTATCCTGATCCCCTCATGCACAACTCTGCTTCAGTATAGCCTAAAACAAGTTTnCCTTTGGCATCA
CAAGCAGTAGGTGTAAAGTCTAGTT
Sample 9, 9 reverse primer
AAGCATATCAGCAGCATGAATAAATTGGTATCCTGATCCCCTCATGCACAACTCTGCTTCAGTATAGCCTAAA
ACAAGTTTTCCTTTGGCATCACAAGCAGTAGGTGTAAAGTCTAGTTTGnGTTTGGTTCTAAAGATGAAATTTT
TGGTTCGGATCTCTATGTCG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript (reverse complement sequence):
CGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGT
GATGCCAAAAGGAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTT
ATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 12, break4 forward primer
TCATGCCACTnTCTCCTGTCTTAATCATCCGGATATGGTACTCAGCACAATAAAGCATATCAGCAGCATGAAT
AAATTGGTATCCTGATCCCCTCATGCACAACTCTGCTTCAGTATAGCCTAAAACAAGTT
Sample 12, 9 reverse primer
AAGCATATCAGCAGCATGAATAAATTGGTATCCTGATCCCCTCATGCACAAnTCTGCTTCAGTATAGCCTAAA
ACAAGTTTTCCTTTGGCATCACAAGCAGTAGGTGTAAAGTCTAGTTTGAGTTTGGTTCTAAAGATGAAATTTT
TGGTTCGGATCTCTATGTCG
47
Transcript detection of the AHR gene in the dog
Combined and corrected the n’s when compared to the already known sequence of the original
transcript (reverse complement sequence):
CGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGT
GATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTT
ATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCATG
A
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 13, break4 forward primer
TCCTGTCTTAATCATCCGGATATGGTACTCAGCACAATAAAGCATATCAGCAGCATGAATAAATTGGTATCCT
GATCCCCTCATGCACAACTCTGCTTCAGTATAGCCTAAAACAAGTTTTCCTTTGGCATCACAAGCAGTAGGT
GTAAAGTCTAGTT
Sample 13, 9 reverse primer
ATAAAGCATATCAGCAGCATGAATAAATTGGTATCCTGATCCCCTCATGCACAAnTCTGCTTCAGTATAGCCT
AAAACAAGTTTTCCTTTGGCATCACAAGCAGTAGGTGTAAAGTCTAGTTTGnGTTTGGTTCTAAAGATGAAAT
TTTTGGTTCGGATCTCTATGTCG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript (reverse complement sequence):
CGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGT
GATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTT
ATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 14, break4 forward primer
TCATGCCACTnnCTCCTGTCTTAATCATCCGGATATGGTACTCAGCACAATAAAGCATATCAGCAGCATGAAT
AAATTGGTATCCTGATCCCCTCATGCACAACTCTGCTTCAGnATAGCCTAAAACAAGTTTTCCTTTGGCATCA
CAAGCAGTAGGTGTAAAGTCTAGTTTGnGTTTG
Sample 14, 9 reverse primer
ATAAAGCATATCAGCAGCATGAATAAATTGGTATCCTGATCCCCTCATGCACAACTCTGCTTCAGTATAGCCT
AAAACAAGTTTTCCTTTGGCATCACAAGCAGTAGGTGTAAAGTCTAGTTTGnGTTTGGTTCTAAAGATGAAAT
TTTTGGTTCGGATCTCTATGTCG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript (reverse complement sequence):
CGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGT
GATGCCAAAGGAAAACTTGTTTTAGGCTATWCTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATT
TATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCAT
GA
Exon 2
48
Transcript detection of the AHR gene in the dog
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 15, break4 forward primer
TCTTAATCATCCGGATATGGTACTCAGCACAATAAAGCATATCAGCAGCATGAATAAATTGGTATCCTGATCC
CCTCATGCACAACTCTGCTTCAGTATAGCCTAAAACAAGTTTnCCTTTGGCATCACAAGCAGTAGGTGTAAA
GTCTAGTT
Sample 15, 9 reverse primer
AAGCATATCAGCAGCATGAATAAATTGGTATCCTGATCCCCTCATGCACAAnTCTGCTTCAGTATAGCCTAAA
ACAAGTTTTCCTTTGGCATCACAAGCAGTAGGTGTAAAGTCTAGTTTGnGTTTGGTTCTAAAGATGAAATTTT
TGGTTCGGATCTCTATGTCG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript (reverse complement sequence):
CGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGT
GATGCCAAAGGAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTA
TTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCAT
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 16, break4 forward primer
CTGTCTTAATCATCCGGATATGGTACTCAGCACAATAAAGCATATCAGCAGCATGAATAAATTGGTATCCTGA
TCCCCTCATGCACAACTCTGCTTCAGTATAGCCTAAAACAAGTTTCCTTTGGCATCACAAGCAGTAGGTGTAAAGTCTAGTT
Sample 16, 9 reverse primer
GCACATAAGCATATCAGCAGCATGAATAAATTGGTATCCTGATCCCCTCATGCACnACTCTGCTTCAGTATAGCCTA
AAACAAGTTTTCCTTTGGCnTCACAAGCAGTAGGTGTAAAGTCTAGTTTGGTTTGGTTCTAAAGATGAAATTTTTGGTTCGGATCTCTATGTCG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript (reverse complement sequence):
CGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACACAACTAGACTTTACACCTACTGCTTG
TGATGCCAAAGGAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTT
ATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAG
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 17, break4 forward primer
49
Transcript detection of the AHR gene in the dog
CATGCCACnTnnTCCTGnCTTAATCATCCGGATATGGTACTCAGCACAATAAAGCATATCAGCAGCATGAATA
AATTGGTATCCTGATCCCCTCATGCACAACnCTGCTTCAGTATAGCCTAAAACAAGTTTCCTTTGGCATCACAAGCAGTAGGTGTAAAGTCTAGTTGTGT
Sample 17, 9 reverse primer
TCAGCAC-ATAAGCATATCAGCAGCATGAATAAATTGGTATCCTGATCCCCTCATGCACAAnTCTGCTTCAGTATAGCCTAAA
ACAAGTTTTCCTTTGGCWTCACAAGCAGTAGGTGTAAAGTCTAGTTTGGTTTGGTTCTAAAGATGAAATTTTTGGTTCGGATCTCTATGTCG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript (reverse complement sequence):
CGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACACAACTAGACTTTACACCTACTGCTTG
TGATGCCAAAGGAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTT
ATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
50
Transcript detection of the AHR gene in the dog
Attachment 2.4 Sequencing results
Exon 10 and 11 of the original transcript
Sample 5, 10 forward primer
CCTAGGTATTGATTTTGAAnATATCAAACACATGCAACAGAATGAGGAAnnnTTCAGAACTGACTTTTCTGGT
GAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCT
GCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCA
CCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAGTGG
AGCAGCAGCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGGAACTCTA
ACCAGTCTGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTGGGAC
CAGTCAGGAGTTTCCCTACAAATCTGAGATTGATGCTATGCCA
Corrected the n’s when compared to the already known sequence of the original transcript:
CCTAGGTATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAATTTTTCAGAACTGACTTTTCTGGT
GAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCT
GCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCA
CCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAGTGG
AGCAGCAGCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGGAACTCTA
ACCAGTCTGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTGGGAC
CAGTCAGGAGTTTCCCTACAAATCTGAGATTGATGCTATGCCA
Exon 10
CAG repeated ‘intron’
Exon 11
Sample 8, 10 forward primer
ACACCTAGGTATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAATnnTTCAGAACTGACTTTTCT
GGTGAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAG
CCTGCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGA
GCACCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAG
TGGAGCAGCAGCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGGAAC
TCTAACCAGTCTGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTG
GGACCAGTCAGGAGTTTCCCTACAAATCTGAGATTGATGCTATGCCATGA
Corrected the n’s when compared to the already known sequence of the original transcript:
ACACCTAGGTATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAATTTTTCAGAACTGACTTTTCT
GGTGAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAG
CCTGCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGA
GCACCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAG
TGGAGCAGCAGCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGGAAC
TCTAACCAGTCTGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTG
GGACCAGTCAGGAGTTTCCCTACAAATCTGAGATTGATGCTATGCCATGA
Exon 10
CAG repeated ‘intron’
Exon 11
Sample 9, 10 forward primer
ATGAAnCnCCTAGGTATTGATTTTGAGATATCAAACACATGCAACAGAATGAGGAAYYTTTCAGAACTGACTTTTCTGGTGAGGATGACTTCAGAGAT
ATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCTGCCTTCGGGTGTTCAGATT
ACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCACCTGCAGTTAGAACAGCAG
CAGCAGCAGCAGCAGC
Corrected the n’s when compared to the already known sequence of the original transcript:
ATGAAACACCTAGGTATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAATTTTTCAGAACTGACT
51
Transcript detection of the AHR gene in the dog
TTTCTGGTGAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAG
TAAGCCTGCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACA
GGAGCACCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGC
Exon 10
CAG repeated ‘intron’
Exon 11
Sample 12, 10 forward primer
CCTAGGTATTGATTTTGAGATATCAAACACATGCAACAGAATGAGGAATTTTTCAGAACTGACTTTTCTGGTG
AGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCTG
CCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCAC
CTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAGTGGA
GCAGCAGCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGnAACTCTAA
CCAGTCTGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTGGGACC
AGTCAGGAGTTTCCnTACAAATCTGAGATTGATGCTATGCCAT
Corrected the n’s when compared to the already known sequence of the original transcript:
CCTAGGTATTGATTTTGAGATATCAAACACATGCAACAGAATGAGGAATTTTTCAGAACTGACTTTTCTGGTG
AGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCTG
CCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCAC
CTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAGTGGA
GCAGCAGCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGGAACTCTAA
CCAGTCTGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTGGGACC
AGTCAGGAGTTTCCCTACAAATCTGAGATTGATGCTATGCCAT
Exon 10
CAG repeated ‘intron’
Exon 11
Sample 13, 10 forward primer
CCTAGGTATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGnnTTTTTCAGAACTGACTTTTCTGGT
GAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCT
GCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCA
CCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAGTGG
AGCAGCAGCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTnGAACTCTA
ACCAGTCTGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTGGGAC
CAGTCAGGAGTTTCCCTACAAATCTGAGATTGATGnTATGCCAT
Corrected the n’s when compared to the already known sequence of the original transcript:
CCTAGGTATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAATTTTTCAGAACTGACTTTTCTGGT
GAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCT
GCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCA
CCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAGTGG
AGCAGCAGCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGGAACTCTA
ACCAGTCTGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTGGGAC
CAGTCAGGAGTTTCCCTACAAATCTGAGATTGATGCTATGCCAT
Exon 10
CAG repeated ‘intron’
Exon 11
Sample 14, 10 forward primer
CCTAGGTATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAnnTTTTCAGAACTGACTTTTCTGGT
GAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCT
GCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCA
CCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAGTGG
AGCAGCAGCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGGAACTCTA
52
Transcript detection of the AHR gene in the dog
ACCAGTCTGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTGGGAC
CAGTCAGGAGTTTCCCTACAAATCTGAGATTGATGCTATGCCAT
Corrected the n’s when compared to the already known sequence of the original transcript:
CCTAGGTATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAATTTTTCAGAACTGACTTTTCTGGT
GAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCT
GCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCA
CCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAGTGG
AGCAGCAGCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGGAACTCTA
ACCAGTCTGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTGGGAC
CAGTCAGGAGTTTCCCTACAAATCTGAGATTGATGCTATGCCAT
Exon 10
CAG repeated ‘intron’
Exon 11
Sample 15, 10 forward primer
TATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAATTTTTCAGAACTGACTTTTCTGGTGAGGAT
GACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCTGCCTTCG
GGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCACCTGCAG
TTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAGTGGAGCAGCA
GCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGGAACTCTAACCAGnC
TGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTGGGACCAGTCAG
GAGTTTCCCTACAAATCTGAGATTGATGCTATGC
Corrected the n’s when compared to the already known sequence of the original transcript:
TATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAATTTTTCAGAACTGACTTTTCTGGTGAGGAT
GACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCTGCCTTCG
GGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCACCTGCAG
TTAGAACAGCAGCAGCAGCAGCAGCAGCAGCTCCTCCAACACCACCAAAATCACATAGCAGTGGAGCAGCA
GCAGCAACTGTGTCAGAAAATGAAGCATATGCAAGTCAATGGCATGTTTGCCAATTGGAACTCTAACCAGTC
TGTGCCTTTTAGTTGTCCTCAGCAAGATCTACAACAGTATAGTGTCTTTTCAGACTTACCTGGGACCAGTCAG
GAGTTTCCCTACAAATCTGAGATTGATGCTATGC
Exon 10
CAG repeated ‘intron’
Exon 11
Sample 17, 10 forward primer
CCTAGGTATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAATTTTTCnGnACTGACTTTTCTGGT
GAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCT
GCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCA
CCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGC
Corrected the n’s when compared to the already known sequence of the original transcript:
CCTAGGTATTGATTTTGAAGATATCAAACACATGCAACAGAATGAGGAATTTTTCAGAACTGACTTTTCTGGT
GAGGATGACTTCAGAGATATTGATATAACAGATGAAATCCTGACATACGTCCAAGATTCTTTAAGTAAGCCT
GCCTTCGGGTGTTCAGATTACCAGCAGCAACAGCCCATGGCTCTGAACTCCAGCTGTATGGTACAGGAGCA
CCTGCAGTTAGAACAGCAGCAGCAGCAGCAGCAGC
Exon 10
CAG repeated ‘intron’
Exon 11
53
Transcript detection of the AHR gene in the dog
Attachment 2.5 Genorm results and meltcurves
qPCR and reference genes
Average expression stability values of remaining control genes
1.4
1.3
Average expression stability M
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
B2M
GAPDH
GUSB
<::::: Least stable genes
HNRPH
RPS5
RPS19
Most stable genes ::::>
Figure 2.5.1 [28]
Determination of the optimal number of control genes for normalization
0.400
0.356
0.350
0.301
0.300
0.250
0.230
0.226
V4/5
V5/6
0.200
0.150
0.100
0.050
0.000
V2/3
V3/4
Pairwise Variations
Figure 2.5.2 [28]
54
Transcript detection of the AHR gene in the dog
Figure 2.5.3 Meltcurve of the original transcript, just one peak is visible, so only one product has
been amplified and measured.
Figure 2.5.4. Meltcurve of the new transcript, just one peak is visible, so only one product has
been amplified and measured.
55
Transcript detection of the AHR gene in the dog
Attachment 2.6 Sequencing results
Length new transcript
Sample 5, break4 forward
AACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAACTTGTTTTAGGCTnTACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGAT
ATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCATGATAGTATTCAGGCTC
CTTACCAAAGACAATCGATGGACCTGGGTTCAGTCTAATGCACGTTTAGTGTATAAAAATGGAAGACCAGAT
TATATCATTGCAACACAGAGACCTCTAACAGATGAAGAAGGAACAGAACATTTACGAAAACGAAATATGAAG
TTGCCTTTTATGTTTACTACTGGAGAAGCTGTGTTGTATGAGATAACAAATCCCTTTCCTCCCATGATGGATC
CCTTACCACTAAGGACTAAAAATGGTGCAAGTGGAAGAGATTCTGCTACCAAATCAACTCTAAATAAGGATT
CTCTCAATCCCAATTCCCTCCTGGCTGCCATGATGCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCAAG
TAGTACACCATTTGAAAGAAATCTTTTTAATGACTCTATGAATGAATGCAGTAATTGGCAAGACAATATCACA
CCCATGGGAAGTGATAGTATCCTAAAACATGAGCAAATAGGnCATTCTCAGGAAATGAATCCAACACTCTCT
GGAGTTCAACCAGGGC
Sample 5, 10 reverse
AAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTT
TTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTT
TATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCATGATAGTATTCAGGCTCCTTACC
AAAGACAATCGATGGACCTGGGTTCAGTCTAATGCACGTTTAGTGTATAAAAATGGAAGACCAGATTATATC
ATTGCAACACAGAGACCTCTAACAGATGAAGAAGGAACAGAACATTTACGAAAACGAAATATGAAGTTGCCT
TTTATGTTTACTACTGGAGAAGCTGTGTTGTATGAGATAACAAATCCCTTTCCTCCCATGATGGATCCCTTAC
CACTAAGGACTAAAAATGGTGCAAGTGGAAGAGATTCTGCTACCAAATCAACTCTAAATAAGGATTCTCTCA
ATCCCAATTCCCTCCTGGCTGCCATGATGCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCAAGTAGTAC
ACCATTTGAAAGAAATCTTTTTAATGACTCTATGAATGAATGCAGTAATTGGCAAGACAATATCACACCCATG
GGAAGTGATAGTATCCTAAAACATGAGCAAATAGGTCA-TCTCAGGAAATGAATCC
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
AAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTT
TTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTT
TATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCATGATAGTATTCAGGCTCCTTACC
AAAGACAATCGATGGACCTGGGTTCAGTCTAATGCACGTTTAGTGTATAAAAATGGAAGACCAGATTATATC
ATTGCAACACAGAGACCTCTAACAGATGAAGAAGGAACAGAACATTTACGAAAACGAAATATGAAGTTGCCT
TTTATGTTTACTACTGGAGAAGCTGTGTTGTATGAGATAACAAATCCCTTTCCTCCCATGATGGATCCCTTAC
CACTAAGGACTAAAAATGGTGCAAGTGGAAGAGATTCTGCTACCAAATCAACTCTAAATAAGGATTCTCTCA
ATCCCAATTCCCTCCTGGCTGCCATGATGCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCAAGTAGTAC
ACCATTTGAAAGAAATCTTTTTAATGACTCTATGAATGAATGCAGTAATTGGCAAGACAATATCACACCCATG
GGAAGTGATAGTATCCTAAAACATGAGCAAATAGGTCATTCTCAGGAAATGAATCCAACACTCTCTGGAGTT
CAACCAGGGC
Exon
Exon
Exon
Exon
7
8
9
10
Sample 8, break4 forward
ACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAACTTGTTTTAGGCTnTACTGAAGCAGAGTTGTGCATGAGGGGATCnGGATACCAATTTATTCATGCTGCTGAT
ATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCnTGATAGTATTCAGGCTC
CTTACCAAAGACAATCGATGGACCTGGGTTCAGTCTAATGCACGTTTAGTGTATAAAAATGGAAGACCAGAT
TATATCATTGCAACACAGAGACCTCTAACAGATGAAGAAGGAACAGAACATTTACGAAAACGAAATATGAAG
TTGCCTTTTATGTTTACTACTGGAGAAGCTGTGTTGTATGAGATAACAAATCCCTTTCCTCCCATGATGGATC
CCTTACCACTAAGGACTAAAAATGGTGCAAGTGGAAGAGATTCTGCTACCAAATCAACTCTAAATAAGGATT
CTCTCAATCCCAATTCCCTCCTGGCTGCCATGATGCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCAAG
TAGTACACCATTTGAAAGAAATCTTTTTAATGACTCTATGAATGAATGCAGTAATTGGCAAGACAATATCACA
56
Transcript detection of the AHR gene in the dog
CCCATGGGAAGTGATAGTATCCTAAAACATGAGCAAATAGGnCATTCTCAGGAAATGAATCC-AACACTCTCTGGAGTTCAACCA
Sample 8, 10 reverse
AAATTTCATCTTTAGAACCAAACACAAnCTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTT
TTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTT
TATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCATGATAGTATTCAGGCTCCTTACC
AAAGACAATCGATGGACCTGGGTTCAGTCTAATGCACGTTTAGTGTATAAAAATGGAAGACCAGATTATATC
ATTGCAACACAGAGACCTCTAACAGATGAAGAAGGAACAGAACATTTACGAAAACGAAATATGAAGTTGCCT
TTTATGTTTACTACTGGAGAAGCTGTGTTGTATGAGATAACAAATCCCTTTCCTCCCATGATGGATCCCTTAC
CACTAAGGACTAAAAATGGTGCAAGTGGAAGAGATTCTGCTACCAAATCAACTCTAAATAAGGATTCTCTCA
ATCCCAATTCCCTCCTGGCTGCCATGATGCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCAAGTAGTAC
ACCATTTGAAAGAAATCTTTTTAATGACTCTATGAATGAATGCAGTAATTGGCAAGACAATATCACACCCATG
GGAAGTGATAGTATCCTAAAACATGAGCAAATAGGTCA-TCTCAGGAAATGAATCCAA
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
AAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTT
TTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTT
TATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCATGATAGTATTCAGGCTCCTTACC
AAAGACAATCGATGGACCTGGGTTCAGTCTAATGCACGTTTAGTGTATAAAAATGGAAGACCAGATTATATC
ATTGCAACACAGAGACCTCTAACAGATGAAGAAGGAACAGAACATTTACGAAAACGAAATATGAAGTTGCCT
TTTATGTTTACTACTGGAGAAGCTGTGTTGTATGAGATAACAAATCCCTTTCCTCCCATGATGGATCCCTTAC
CACTAAGGACTAAAAATGGTGCAAGTGGAAGAGATTCTGCTACCAAATCAACTCTAAATAAGGATTCTCTCA
ATCCCAATTCCCTCCTGGCTGCCATGATGCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCAAGTAGTAC
ACCATTTGAAAGAAATCTTTTTAATGACTCTATGAATGAATGCAGTAATTGGCAAGACAATATCACACCCATG
GGAAGTGATAGTATCCTAAAACATGAGCAAATAGGTCATTCTCAGGAAATGAATCC-AACACTCTCTGGAGTTCAACCA
Exon
Exon
Exon
Exon
7
8
9
10
Sample 9, break4 forward
AACAC-AACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCT
GATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCnTGATAGTATTCAGG
CTCCTTACCAAAGACAATCGATGGACCTGGGTTCAGTCTAATGCACGTTTAGTGTATAAAAATGGAAGACCA
GATTATATCATTGCAACACAGAGACCTCTAACAGATGAAGAAGGAACAGAACATTTACGAAAACGAAATATG
AAGTTGCCTTTTATGTTTACTACTGGAGAAGCTGTGTTGTATGAGATAACAAATCCCTTTCCTCCCATGATGG
ATCCCTTACCACTAAGGACTAAAAATGGTGCAAGTGGAAGAGATTCTGCTACnAAATCAACTCTAAATAAGG
ATTCTCTCAATCCCAATTCCCTCCTGGCTGCCATGATGCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCA
AGTAGTACACCATTTGAAAGAAATCTTTTTAATGACTCTATGAATGAATGCAGTAATTGGCAAGACAATATCA
CACCCATGGGAAGTGATAGTATCCTAAAACATGAGCAAATAGGTCATTCTCAGGAAATGAATCnAACA
Sample 9, 10 reverse
CTnGnCTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCA
TGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGA
TTAAGACAGGAGAGAGTGGCATGATAGTATTCAGGCTCCTTACCAAAGACAATCGATGGACCTGGGTTCAG
TCTAATGCACGTTTAGTGTATAAAAATGGAAGACCAGATTATATCATTGCAACACAGAGACCTCTAACAGATG
AAGAAGGAACAGAACATTTACGAAAACGAAATATGAAGTTGCCTTTTATGTTTACTACTGGAGAAGCTGTGT
TGTATGAGATAACAAATCCCTTTCCTCCCATGATGGATCCCTTACCACTAAGGACTAAAAATGGTGCAAGTG
GAAGAGATTCTGCTACCAAATCAACTCTAAATAAGGATTCTCTCAATCCCAATTCCCTCCTGGCTGCCATGAT
GCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCAAGTAGTACACCATTTGAAAGAAATCTTTTTAATGACT
CTATGAATGAATGCAGTAATTGGCAAGACAATATCACACCCATGGGAAGTGATAGTATCCTAAAACATGAGC
AAATAGGTCA-TCTCAGGAAATGAATCCAACA
57
Transcript detection of the AHR gene in the dog
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
AACACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAG
AGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATAT
CCGGATGATTAAGACAGGAGAGAGTGGCATGATAGTATTCAGGCTCCTTACCAAAGACAATCGATGGACCT
GGGTTCAGTCTAATGCACGTTTAGTGTATAAAAATGGAAGACCAGATTATATCATTGCAACACAGAGACCTC
TAACAGATGAAGAAGGAACAGAACATTTACGAAAACGAAATATGAAGTTGCCTTTTATGTTTACTACTGGAG
AAGCTGTGTTGTATGAGATAACAAATCCCTTTCCTCCCATGATGGATCCCTTACCACTAAGGACTAAAAATGG
TGCAAGTGGAAGAGATTCTGCTACCAAATCAACTCTAAATAAGGATTCTCTCAATCCCAATTCCCTCCTGGCT
GCCATGATGCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCAAGTAGTACACCATTTGAAAGAAATCTTT
TTAATGACTCTATGAATGAATGCAGTAATTGGCAAGACAATATCACACCCATGGGAAGTGATAGTATCCTAA
AACATGAGCAAATAGGTCATTCTCAGGAAATGAATCCAACA
Exon
Exon
Exon
Exon
7
8
9
10
Sample 11, break4 forward
AACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGT
GCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGA
TGATTAAGACAGGAGAGAGTGGCnTGATAGTATTCAGGCTCCTTACCAAAGACAATCGATGGACCTGGGTT
CAGTCTAATGCACGTTTAGTGTATAAAAATGGAAGACCAGATTATATCATTGCAACACAGAGACCTCTAACA
GATGAAGAAGGAACAGAACATTTACGAAAACGAAATATGAAGTTGCCTTTTATGTTTACTACTGGAGAAGCT
GTGTTGTATGAGATAACAAATCCCTTTCCTCCCATGATGGATCCCTTACCACTAAGGACTAAAAATGGTGCAA
GTGGAAGAGATTCTGCTACCAAATCAACTCTAAATAAGGATTCTCTCAATCCCAATTCCCTCCTGGCTGCCAT
GATGCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCAAGTAGTACACCATTTGAAAGAAATCTTTTTAATG
ACTCTATGAATGAATGCAGTAATTGGCAAGACAATATCACACCCATGGGAAGTGATAGTATCCTAAAACATG
AGCAAATAGGTCATTCTCAGnAAATGAATCCAACACTCTCTGGA
Sample 11, 10 reverse
ACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGA
TCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAG
GAGAGAGTGGCATGATAGTATTCAGGCTCCTTACCAAAGACAATCGATGGACCTGGGTTCAGTCTAATGCA
CGTTTAGTGTATAAAAATGGAAGACCAGATTATATCATTGCAACACAGAGACCTCTAACAGATGAAGAAGGA
ACAGAACATTTACGAAAACGAAATATGAAGTTGCCTTTTATGTTTACTACTGGAGAAGCTGTGTTGTATGAGA
TAACAAATCCCTTTCCTCCCATGATGGATCCCTTACCACTAAGGACTAAAAATGGTGCAAGTGGAAGAGATT
CTGCTACCAAATCAACTCTAAATAAGGATTCTCTCAATCCCAATTCCCTCCTGGCTGCCATGATGCAACAAGA
TGAGTCTATTTATCTCTATCCTTCCTCAAGTAGTACACCATTTGAAAGAAATCTTTTTAATGACTCTATGAATG
AATGCAGTAATTGGCAAGACAATATCACACCCATGGGAAGTGATAGTATCCTAAAACATGAGCAAATAGGTC
A-TCTCAGGAAATGAATCC-ACA
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
AACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGT
GCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGA
TGATTAAGACAGGAGAGAGTGGCATGATAGTATTCAGGCTCCTTACCAAAGACAATCGATGGACCTGGGTT
CAGTCTAATGCACGTTTAGTGTATAAAAATGGAAGACCAGATTATATCATTGCAACACAGAGACCTCTAACA
GATGAAGAAGGAACAGAACATTTACGAAAACGAAATATGAAGTTGCCTTTTATGTTTACTACTGGAGAAGCT
GTGTTGTATGAGATAACAAATCCCTTTCCTCCCATGATGGATCCCTTACCACTAAGGACTAAAAATGGTGCAA
GTGGAAGAGATTCTGCTACCAAATCAACTCTAAATAAGGATTCTCTCAATCCCAATTCCCTCCTGGCTGCCAT
GATGCAACAAGATGAGTCTATTTATCTCTATCCTTCCTCAAGTAGTACACCATTTGAAAGAAATCTTTTTAATG
ACTCTATGAATGAATGCAGTAATTGGCAAGACAATATCACACCCATGGGAAGTGATAGTATCCTAAAACATG
AGCAAATAGGTCATTCTCAGGAAATGAATCCAACACTCTCTGGA
Exon
Exon
Exon
Exon
7
8
9
10
58
Transcript detection of the AHR gene in the dog
Attachment 2.7 Sequencing results
Confirmation new transcript
Sample 5, break1 forward
CTAGACTTTACACCTACTGCTTGnGATGCCAAAGGAAAACnTnTTTTAGGCTATACTGAAGCAGAGTTGTGCA
TGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGnGCTGAGTACCATATCCGGATGA
TTAAGACAGGAnAGAGTGGCATnA
Sample 5, 9 reverse
TTCCAAGCGACnTAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGnGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATATG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGA
GTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 8, break1 forward
ACC-AACAC—
AACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTnTTTTAGGCTATACTGAAGCAGAGTTGTG
CATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGAT
GATTAAGACAGGAnAnAGTGGCATGA
Sample 8, 9 reverse
TTCCAAGCGACnTAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCAnGnGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATATGCTT-A-TGTGCTGA
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGA
GTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 9, break1 forward
CAC—
AACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAnCnTnTTTTAGGCTATACTGAAGCAGAGTTGTG
59
Transcript detection of the AHR gene in the dog
CATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGAT
GATTAAGACAGGAGAGAGTGGCATGA
Sample 9, 9 reverse
TTCCAAGCGAnnTAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGnCTATACTGAAGCAGAGTTGTGCAnGnGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATATGCTT-A-TGTGCT
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGA
GTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 11, break1 forward
ACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAnCnTnTTTTAGGCTATACTGAAGCAGAGTT
GTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCG
GATGATTAAGACAGGAnAGAGTGGCATnA
Sample 11, 9 reverse
TTCCAAGCGAnnTAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGnAnnnGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATATG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGA
GTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 12, break1 forward
AACACAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGT
GCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGA
TGATTAAnACAGGAnAnAGTGGCATnA
Sample 12, 9 reverse
TTCCAAGCGnnnTAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCTTGAGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATATGCTT-A
60
Transcript detection of the AHR gene in the dog
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGA
GTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 13, break 1 forward
CTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAnCTTGTTTTAGGCTATACTGAAGCAGAGTTGTGC
ATGAGGGGnTCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATG
ATTAAGACAGGAnAnAGTGGCATnA
Sample 13, 9 reverse
TTCCAAGCGAnnTAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGnGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATATG
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGA
GTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 5, break2 forward
failed
Sample 5, 9 reverse
TTnCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCAnG
Corrected the n’s when compared to the already known sequence of the original transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATG
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
61
Transcript detection of the AHR gene in the dog
Exon 9
The T belongs to exon 7
Sample 8, break2 forward
CTTnTTTTAGGCTATACTGAAGCAnAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATA
TGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGTGGCATnA
Sample 8, 9 reverse
TTnCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCAnGAGGGGATCAGGAT
A
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGA
GTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 9, break2 forward
TTTTAGGCTATACTGAAGCAnAGTTGTGCATGAGGGGATCAGGAnACCAATTTATTCnTGCTGCTGATATGCT
TTATTGTGCTGAGTACCATATCCGGATGATTAAGAC
Sample 9, 9 reverse
TTYCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCAnGAGGGGATCAGGAT
A
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGAC
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 11, break2 forward
AACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAnAGTTGTG
CATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGAT
GATTAAnACAGGAnAnAGTGGCATnA
Sample 11, 9 reverse
62
Transcript detection of the AHR gene in the dog
TTnCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCT
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGA
GTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 12, break2 forward
CTGCTnnTGATGCCAAAGGAAAACTTnTTTTAGGCTATACTGAAGCAnAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGnGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGA
GTGGCATnA
Sample 12, 9 reverse
TTnCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCnAnTTATTCATGC
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TTCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTAC
TGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGAT
ACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGA
GTGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 13, break2 forward
GGCTATACTGAAGCAnAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTAT
TGTGCTGAGTACCATATCCGGATGATTAAGACAGGAnAnAGTGGCATnA
Sample 13, 9 reverse
TnCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACT
GCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATA
nCnAnTTATTCATGCTGCTGATA-G
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACT
GCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATA
63
Transcript detection of the AHR gene in the dog
CCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAG
TGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 5, break3 forward
AACTAGACTTTACACCTACTGCTTGTGATGCCAAAGnAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTG
CATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGAT
GATTAAGACAGGnnAGAGTGGCATGAT
Sample 5, 9 reverse
TCCAAGCGACATAnnGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACT
GCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATA
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACT
GCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAG
TGGCATGAT
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 8, break3 forward
A-C-AACACAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGT
GCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGA
TGATTAAGACAGGnGAnAGTGGCATGA
Sample 8, 9 reverse
TCCAAGCGACATAGnnATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACT
GCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCAnGAGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATAT
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACT
GCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAG
TGGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
64
Transcript detection of the AHR gene in the dog
The T belongs to exon 7
Sample 9, break3 forward
AACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGT
GCATGAGGGGATCAGGATACCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGA
TGATTAAGACAGGnGAGAGTGGCATGA
Sample 9, 9 reverse
CCAAGCGACATAnAnATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTG
CTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATAC
CAATTTATTCATGCTGCTGATATGCTT-A
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
CCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACTG
CTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATAC
CAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAGT
GGCATGA
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
Sample 11, break3 forward
A-CA
ACACAAACTAGACTTTACACCTACTGCTTGTGATGCCAAAGGAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATACCAATTTATTCATGCTGCT
GATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGG-GAnAGTGGCATGAT
Sample 11, 9 reverse
TCCAAGCGACATAGnGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACT
GCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATA
CCAATTTAT-CATGCTGCTGATATGCTT-A-TGTGCTGA
Combined and corrected the n’s when compared to the already known sequence of the original
transcript:
TCCAAGCGACATAGAGATCCGAACCAAAAATTTCATCTTTAGAACCAAACACAAACTAGACTTTACACCTACT
GCTTGTGATGCCAAAGGAAAACTTGTTTTAGGCTATACTGAAGCAGAGTTGTGCATGAGGGGATCAGGATA
CCAATTTATTCATGCTGCTGATATGCTTTATTGTGCTGAGTACCATATCCGGATGATTAAGACAGGAGAGAG
TGGCATGAT
Exon 2
Overlap exon 2 and 7
Exon 7
Exon 8
Exon 9
The T belongs to exon 7
65