Download Prof. Kamakaka`s Lecture 14 Notes

Studying differences/similarities in Individuals
Methods used to study differences between individuals
RFLP
SNP
DNA Repeats
1
Ind1
ATTGTATTGATTTATAGCGCGCGCGCTAGTTGACTGG
Ind2
ATTGTATTGATTTATAGCGCGCTAGTTGACTGG
Ind3
ATTCTATTGATTTATAGCGCGCGCGCTAGTTGACTGG
2
Genetic polymorphism
•Genetic Polymorphism: A difference in DNA sequence among
individuals, groups, or populations.
Genetic mutations are a kind of genetic polymorphism.
Genetic Variation
Single nucleotide
Polymorphism
(point mutation)
Repeat heterogeneity
Ind1
ATTGTATTGATTTATAGCGCGCGCGCTAGTTGACTGG
Ind2
ATTGTATTGATTTATAGCGCGCTAGTTGACTGG
Ind3
ATTCTATTGATTTATAGCGCGCGCGCTAGTTGACTGG
3
Repeats and DNA fingerprint
Variation between people- small DNA change – a single
nucleotide polymorphism [SNP] – in a target site,
RFLPs and point mutations are proof of variation at the DNA
level.
Satellite sequences: a short sequence of DNA repeated many
times.
Chr1
Interspersed
Chr2
tandem
4
Repeats
Satellite (large) (100->1000 bps long) Centromere/telomere
Micro-satellite Extremely small repeats (2-5bp)GCGCGCGC
AGCAGCAGCAGC
Mini-satellite- larger repeats (6-100 bp long)
Repeats can be dispersed or in tandem-
E
E
2
E
5
E
6
Chr1
Chr2
3
E
1
E
4
tandem
E
0.5
E
5
Mini Satellite Repeats and Blots
Mini Satellite sequences: a short sequence (20-100bp long) of
DNA repeated many times
E
E
2
E
5
E
6
Chr1
Chr2
3
1
E
E
M
E tandem
DNA
7
4
M
Take Genomic
DNA, Digest with
EcoRI, Probe
southern blot with
repeat probe
5
3
3
1
1
DNA gel
E
DNA
7
5
0.5
Blot
6
Mini Satellite Repeats and Blots
Mini Satellite sequences: a short sequence (20-100bp long) of
DNA repeated many times
E
E
2
E
5
E
6
Chr1
Chr2
3
1
E
E
M
E
DNA
7
4
tandem
M
E
DNA
Take Genomic
DNA, Digest with
EcoRI, Probe
southern blot with
repeat probe
7
5
5
3
3
1
1
DNA gel
0.5
Blot
7
Repeat expansion/contraction
Tandem repeats expand and contract during recombination.
Mistakes in pairing leads to changes in tandem repeat numbers
These can be detected by Southern blotting because as the
number of repeats expand at a specific site, the restriction
fragment at that site expands in size
An allele of a mini-satellite varies by the number of repeats
One repeat to many repeats- (varying in length from 0.5 to 20
kb)
Chr1 Individual 1
4
E
E
Chr1 Individual 2
5
E
Ind2
Ind1
E
5
3
There are on average
between 2 and 10 alleles
(repeats) per mini-sat locus
1
8
Ind3
Ind2
Ind1
Homozygous/heterozygous?
5
3
1
Chr1
E
E
Chr1
E
E
9
Micro-satellite and PCR
Microsatellite repeat expansion and contraction is
investigated using PCR and gels instead of gels and
southern blots
AGCGTCAGCGCGCGCTTATTGA
TCGCAGTCGCGCGCGAATAACT
AGCGTCA
AATAACT
22 bp PCR fragment
AGCGTCAGCGCGCGCGCGCGCTTATTGA
TCGCAGTCGCGCGCGCGCGCGAATAACT
AGCGTCA
AATAACT
28 bp PCR fragment
10
GC GC
GC GC
GC
GC GC
GC
TA TA
TA
TA
TA TA
GC
2
GC
1
GC
2
GC
1
GC GC
TA
TA
Genotype
Locus1
Individual1
allele5, allele2
Individual2
allele4, allele3
Locus2
allele2, allele1
allele3, allele2
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Microsatellite DNA fingerprint
1 2 3 4
1
2
In this example, 2 totally different microsatellites (1) and (2)
located on the short arm of chromosome 6 have been amplified
by the polymerase chain reaction (PCR).
The PCR products are labeled with a blue or green fluorescent
marker and run in a gel each lane showing the genetic profile of
a different individual.
Each individual has a different genetic profile because each
person has a different number of microsatellite length repeats,
the number of repeats giving rise to bands of different sizes
after PCR.
Locus1
locus2
Indi1
allele2,allele4
allele6,allele6
Indi2
allele2, allele6
allele1, allele4
Indi3
allele5, allele6
allele4, allele5
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Crossover in mispaired duplicated segments
What happens if you get a crossover after mis-pairing in
meiosisI?
B
C
A
B
C
D
E
a
b
c
d
e
b
c
A
B
C
B
C
D
A
B
C
B
c
b
a
b
C
D
a
b
c
b
E
c
d
e
E
c
d
e
13
FBI and Microsatellite
The FBI uses a set of 13 different microsatellite markers in
forensic analysis.
13 sets of specific PCR primers are used to determine the allele
present in the test sample for each marker.
The marker used, the number of alleles at each marker and the probability
of obtaining a random match for a marker is shown.
How often would you expect an individual to be mis-identified if all 13
markers are analyzed
Locus
No. of alleles
A
B
C
D
E
F
G
H
I
J
K
L
M
11
19
7
7
10
10
10
11
10
8
8
15
20
probability of
random match
0.112
0.036
0.081
0.195
0.062
0.075
0.158
0.065
0.067
0.085
0.089
0.028
0.039
P= 0.112x0.036x0.081x0.195x----- =
= 1.7x10-15
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Variation between people- small DNA change – a single nucleotide
polymorphism [SNP] – in a target site,
RFLPs and SNPs are proof of variation at the DNA level,
Satellite sequences: a short sequence of DNA repeated many
times.
Micro satellite are 2-5 bp repeats in tandem repeats 15-100
times in a row
Mini satellite are 6-100 bp repeats in tandem (0.5 to 20kb
long)
Class
size
No of loci
method
Micro
~200bp
200,000
PCR
Mini
0.2-20kb
30,000
southern blot
SNP
1 bp
100 million
PCR/microarray
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DNA Fingerprint Analysis
Mr. Chan’s family consists of mom, dad and four kids. The
parents have one daughter and one son together, another
daughter is from the mother’s previous marriage, and the
other son is adopted. Here are the DNA analysis results:
1. Which child is adopted?
Child4
1. Which child is from the mother’s previous
marriage?
Child2
1. Who are the own children of Mr and Mrs
Chan?
Child1 and Child3
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Individuals
Methods used to study differences between individuals
DNA Repeats
RFLP
SNP
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Very small Deletion (of a restriction site)
1kb
2kb
E
E
3kb
4kb
GeneR
GeneC
8kb
H
Marke
r
Marke
r
3kb
EcoRI
WT
E
5kb
4.5kb
GeneX
H
E
0.5kb
GeneA
9kb
Marke
r
E
Marke
r
E
EcoRI
Deletion
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RFLP analysis
RFLP= Restriction fragment length polymorphism
Refers to variation (presence or absence) in restriction sites
between individuals Because of mutations in Restriction sites
These are extremely useful and valuable for geneticists (and
lawyers)
On average two individuals (humans) vary at 1bp in every 3001000 bp
The human genome is 3x109 bp
This means that they will differ in more than 3 million bp!!!
By chance these changes will CREATE or DESTROY the
recognition sites for restriction enzymes
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RFLP
Lets generate a EcoRI map for the region in one individual
3kb
GAATTC
4kb
GAATTC
GAATTC
The the same region of a second individual may appear as
The nucleotides are not deleted – just changed
7kb
GAGTTC
GAATTC
1
Normal
GAATTC
Mutant
GAGTTC
2
EcoRI
Marker
GAATTC
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RFLP
The internal EcoRI site is missing in the second individual
For X1 the sequence at this site is GAATTC
CTTAAG
This is the sequence recognized by EcoRI
The equivalent site in the X2 individual is different
GAGTTC
CTCAAG
This sequence IS NOT recognized by EcoRI and is therefore
not cut
Now if we examine a large number of humans at this site we
may find that 25% possess the EcoRI site and 75% lack this
site.
We can say that a restriction fragment length polymorphism
exits in this region
These polymorphisms usually do not have any phenotypic
consequences
Silent mutations that do not alter the protein sequence
because of redundancy in codon usage, localization in introns
or non-genic regions
21
RFLP
RFLP are identified by southern blots
In the region of the human X chromosome, two forms of the
X-chromosome are Segregating in the population.
4
B
5
R
3
R
6
R
X1
3.5
B
1
R
2
Digest DNA with
EcoRI or BamHI and
probe with
Probe1/ probe2
What do we get?
8
4
B
R
6
R
1
X2
3.5
B
R
2
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RFLP in individuals
If we used probe1 for southern blots with a BamHI digest what
would be the results for X1/X1, X1/X2 and X2/X2 individuals?
X1/X1
Probe1
X1/X2
X2/X2
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BamHI
18
18
If we used probe1 for southern blots with a EcoRI digest what
would be the results for X1/X1, X1/X2 and X2/X2 individuals?
X1/X1
Probe1
Probe2
B
5
R
4
B
9.5
6
R
9.5
X1
3.5
B
8
R
2
8
R
8, 5, 3
9.5
3
R
1
X2/X2
5, 3
EcoRI
EcoRI
4
X1/X2
R
1
6
R
X2
3.5
B
R
2
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RFLP
RFLP’s are found by trial and error
They require an appropriate probe and appropriate enzyme
They are very valuable because they can be used just like any
other genetic marker to map genes
They are employed in recombination analysis (mapping) in the
same way as conventional morphological allele markers are
employed
The presence of a specific restriction site at a specific locus on
one chromosome and its absence at a specific locus on another
chromosome can be viewed as two allelic forms of a gene
The phenotype in this case is a Southern blot rather than white
eye/red eye
4
6
R
2
R
X1
2
R
1
R
R
4
5
R
2
1
4
8
R
R
1
X2
2
R
R
1
R
4
R
2
R
3
242
R
4
6
R
2
R
X1
2
R
1
R
R
4
5
R
2
1
4
8
R
R
1
X2
2
R
R
Probe1
1
R
4
R
2
R
3
2
Probe2
6+2
X1/X1 individual
5
8
X2/X2 individual
3
25
R
RFLPs can be followed in pedigrees
1
Alleles
3
C
7
4
GAGTTC
A
3
4
4
R1
CA
CC
AA
CA
R3R3+
R3R3-
R3+
R3+
R3R3+
R3
GAATTC
Mutation is recessive
Most likely Resides at or near R3-
R4
RFLPs can be followed in pedigrees
Alleles
8
6
C
2
2
4
B
2
2
4
A
2
2
4
4
R1
CA
CB
AA
R2
BA
R2-R3- R2-R3- R2+R3+ R2+R3R2+R3+ R2+R3- R2+R3+ R2+R3+
Mutation is recessive
Most likely Resides at or near R3
R3
R4
Using RFLPs to map human disease genes
8 EcoRI
6 Probe3
5 EcoRI
3 Probe2
9 EcoRI
1 Probe1
1
1
2
2
3
3
Which RFLP pattern segregates specifically with all of the
diseased individual BUT NOT WITH NORMAL INDIVIDUALS?
1, 2 or 3
Which band segregates with the phenotype
Top or bottom?
Using DNA probes for different RFLPs you can screen
individuals for a RFLP pattern that shows co-inheritance with
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the disease
Conclusion: the actual mutation resides at or near RFLP3
bottom band (Dominant ?)
Mapping
To map Two Genes, you perform crosses and measure
recombination frequency between the two genes IN A
HETEROZYGOUS INDIVIDUAL.
Gene W and B are responsible for wing and bristle development
W
Centromere
B
Telomere
To find the map distance between these two genes we need
ALLELIC variants at each locus
W=wings
w= No wings
B=Bristles
b= no bristles
To measure distance between these two genes, You do a TEST
CROSS
A double heterozygote is crossed to the double homozygote
recessive
WB/wb
Female
Wings
Bristles
X
wb/wb
Males
No wings
No bristles
----W--------B------w--------b---
----w--------b------w--------b---
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Mapping
WB/wb
Female
Wings
Bristles
X
wb/wb
Males
No wings
No bristles
Female gamete
Male gamete (wb)
Genotype
phenotype
WB
WB/wb
Wings
Bristles
51
wb
wb/wb
No wings
No bristles
43
Wb
Wb/wb
Wings
No bristles
3
wB
wB/wb
No wings
Bristles
4
Map distance= # recombinants /Total progeny
7/101= 7 M.U.
30
Mapping
To find the map distance between genes, multiple alleles are
required to measure appearance of recombinants
We know the distance between W and B by the classical method
because multiple alleles exist at each locus
(W & w, B & b). It is 7MU.
We know the distance between B and R by the classical method as
20MU by using heterozygotes for B and R in a cross and looking
for recombinants
7MU
Centromere
W
20MU
B
C
R
Telomere
Now suppose you find a new gene C.
You could map this gene with respect to Genes W, B and R using
classical methods.
However, what if it is difficult to study the function of this new
gene (the phenotype is difficult to see with the naked eye)
If the researcher identifies an RFLP in this gene you can 31
map
the gene mutation by simply following the RFLP.
Mapping
Prior to RFLP analysis, only a few classical gene markers
existed (approximately 200 in humans)
Now over 7000 RFLPs have been mapped in the human genome.
Newly inherited disorders are now mapped by determining
whether they are linked to previously identified RFLPs
7MU
Centromere
W
or
w
Centromere RFLP1
Probe1
7kb
or
4kb
20MU
B
or
b
R
or
r
Telomere
RFLP2
RFLP3
Telomere
Probe2
1kb
or
2kb
Probe3
3kb
or
9kb
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RFLP Mapping
Both the normal and mutant alleles of gene B (B and b) are
sequenced and we find
W
Centromere
B
B
GAATTC
3
2
E
Telomere
E
E
b
E
5
E
AAATTC
The mutation disrupts the sequence and alters a EcoRI site!
If DNA is isolated from B/B, B/b and b/b individuals, cut
with EcoRI and probed in A Southern blot, the pattern that
we will obtain is
B/B Bristle
B/b Bristle
b/b No bristle
33
Mapping
To find the map distance between two genes we need
ALLELIC variants at each locus
Therefore in the cross (WB/wb x wb/wb), the genotype
at the B locus can be distinguished either by the presence and
absence of bristles OR by a Southern blot
WB/wb
Female
x
wb/wb
Male
Wings
Bristles
No wings
No Bristles
Southern blot:
Southern blot:
5 and 2 kb band
5 kb band
You can use RFLPs instead of genes as markers along a
chromosome
Just like Genes, RFLPs mark specific positions on chromosomes
and can be used for mapping.
34
Mapping
Female gamete
Male gamete (wb)
Parental
Genotype
phenotype
WB
WB/wb
Wings
RFLP 5kb 2kb
51
wb
wb/wb
No wings
RFLP 5kb 5kb
43
Wb
Wb/wb
Wings
RFLP 5kb 5kb
3
wB
wB/wb
No wings
RFLP 5kb 2kb
4
Recombinant
35
Map distance= # recombinants /Total progeny 7/101= 7 M.U.
Individuals
Methods used to study differences between individuals
RFLP
SNP
DNA Repeats
36
Genetic polymorphism
•Genetic Polymorphism: A difference in DNA sequence among
individuals, groups, or populations.
•Genetic Mutation: A change in the nucleotide sequence of a
DNA molecule.
Genetic mutations are a subset of genetic polymorphism
Genetic Variation
Single nucleotide
Polymorphism
(point mutation)
Repeat heterogeneity
37
SNP
•A Single Nucleotide Polymorphism is a source variance in a
genome.
•A SNP ("snip") is a single base change in DNA.
•SNPs are the most simple form and most common source of
genetic polymorphism in the human genome (90% of all human
DNA polymorphisms).
•There are two types of nucleotide base substitutions resulting
in SNPs:
–Transition: substitution between purines (A, G) or
between pyrimidines (C, T). Constitute two thirds of all
SNPs.
–Transversion: substitution between a purine and a
pyrimidine.
While a single base can change to all of the other three
bases, most SNPs have only one allele.
38
SNPs-
Single Nucleotide Polymorphisms
-----------------------ACGGCTAA
-----------------------ATGGCTAA
Instead of using restriction enzymes, these are found by
direct sequencing/PCR
They are extremely useful for mapping
Markers
Classical Mendelian
RFLPs
SNPs
~200
7000
1.4x106
SNPs occur every 300-1000 bp along the 3 billion long human
genome
Many SNPs have no effect on cell function
Note: RFLPs are a subclass of SNPs
39
SNPs
Humans are genetically >99 per cent identical: it is the
tiny percentage that is different
Much of our genetic variation is caused by single-nucleotide
differences in our DNA : these are called single nucleotide
polymorphisms, or SNPs.
As a result, each of us has a unique genotype that typically differs in
about three million nucleotides from every other person.
SNPs occur about once every 300-1000 base pairs in the genome, and
the frequency of a particular polymorphism tends to remain stable in
the population.
Because only about 3 to 5 percent of a person's DNA sequence codes
for the production of proteins, most SNPs are found outside of
"coding sequences".
40
How did SNPs arise?
F2a----ACGGACTGAC----CCTTACGTTG----TACTACGCAT---|
F1 ----ACTGACTGAC----CCTTACGTTG----TACTACGCAT---P
----ACTGACTGAC----CCTTACGTTG----TACTACGCAT----
|
F1 ----ACTGACTGAC----CCTTACGTTG----TACTAGGCAT---|
|
F2b----ACTGACTGAC----CCATACGTTG----TACTAGGCAT----
Compare the two F2 progeny
Haplotype1 (F2a) = SNP allele1
----ACGGACTGAC----CCTTACGTTG----TACTACGCAT---Haplotype2 (F2b) = SNP allele2
----ACTGACTGAC----CCATACGTTG----TACTAGGCAT----
41
Each of 1013 cells in the human body receives approximately
thousand DNA lesions per day (Lindahl and Barnes 2000)
When these mutations are not repaired they are fixed in the
genome of that particular cell
If a mutation is fixed in germ cells that go on to be fertilized
and form an embryo they will be propagated to progeny
It was calculated that there are ~70 new chnages in each
diploid human genome
42
SNPs, RFLPs, point mutations
GAATTC
GAATTC
GAATTC
GAATTC
GAGTTC
GAATTC
RFLP
SNP
SNP
Pt mut
SNP
GAATTC
GAATTC
GAATTC
GACTTC
RFLP
Pt mut
SNP
43
Coding Region SNPs
•Types of coding region SNPs
–Synonymous: the substitution causes no amino acid change to
the protein it produces. This is also called a silent mutation.
–Non-Synonymous: the substitution results in an alteration of
the encoded amino acid. A missense mutation changes the
protein by causing a change of codon. A nonsense mutation
results in a misplaced termination.
–More than half of all coding sequence SNPs result in
non-synonymous codon changes.
Intergenic SNPs
Researchers have found that most SNPs are not responsible for a
disease state because they are intergenic SNPs
Instead, they serve as biological markers for pinpointing a disease on
the human genome map, because they are usually located near a gene
found to be associated with a certain disease.
Scientists have long known that diseases caused by single genes and
inherited according to the laws of Mendel are actually rare.
Most common diseases, like diabetes, are caused by multiple genes.
Finding all of these genes is a difficult task.
Recently, there has been focus on the idea that all of the genes
involved can be traced by using SNPs.
By comparing the SNP patterns in affected and non-affected
individuals—patients with diabetes and healthy controls, for
example—scientists can catalog ALL of the DNA sequence variations
in affected Vs unaffected individuals to identify mutations that
underlie susceptibility for diabetes
GAATTC
GAGTTC
GAATTC
RFLP
SNP
SNP
Pt mut
SNP
GAATTC
GACTTC
RFLP
Pt mut
SNP
PCR
If a region of DNA has already been sequenced in one individual,
the sequence information can be used to isolate and amplify that
sequence from other individuals DNA in a population.
Individuals with mutations in p53 are at risk for colon cancer
To determine if an individual had such a mutation, prior to PCR
one would have to clone the gene from the individual of interest
(construct a genomic library, screen the library, isolate the clone
and sequence the gene).
With PCR, the gene can be isolated directly from DNA isolated
from that individual.
No lengthy cloning procedure necessary
Only small amounts of genomic DNA required
30 rounds of amplification can give you >109 copies of a gene
46
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
PRIMER1 5’AAAGATC3’
3’AGCTAGAT5’ PRIMER2
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’AGCTAGAT5’
WT
5’AAAGATC3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
Agarose
Gel
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
47
SNPs and Primers- ASO hybridization
Individual 1
GACTCCTGAGGAGAAGTG
Individual 2
GACTCCTGTGGAGAAGTG
Raise Temperature
Raise Temperature
2
1
DNA from individuals 1 and 2 are tested under
CONDITIONS that only allow perfect matches of oligos to
anneal to the genomic DNA.
48
Mutants cannot be amplified
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
PRIMER1 5’AAAGATC3’
3’AGCTAGAT5’ PRIMER2
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’AGCTAGAT5’
WT
5’AAAGATC3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3‘TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
Agarose
Gel
49
Mutants cannot be amplified
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
PRIMER1 5’AAAGATC3’
3’AGCTAGAT5’ PRIMER2
5’AAAGATCGGGGGGGGGGGGGGGTCGATTTA3’
3’AGCTAGAT5’
mut
WT
5’AAAGATC3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’AGCTAGAT5’
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
Agarose
Gel
50
PCR allows only one SNP to be tested at a time
1
aAa
2
cCc
3
A
4
G
5
G
6
T
7
A
8
T
9
C
Ind1
aTa
cGc
A
T
G
T
G
T
G
Ind2
Oligonucleotide chips contain thousands of short DNA
sequences immobilised at different positions. Such chips can
be used to discriminate between alternative bases at the site
of a SNP.
Chips allow many SNPs to be analyzed in parallel.
Short DNA sequences on the chip represent all possible
variations at a polymorphic site;
A labeled genomic DNA from an individual will only stick if
there is an exact match. The base is identified by the location
of the fluorescent signal.
51
Microarrays and SNPs
SNP1 B
SNP1 A
GACTCCTGTGGAGAAGTG
GACTCCTGAGGAGAAGTG
SNP2 B
SNP2 A
GGGGGGGGCGGGGGGGGG
GGGGGGGGGGGGGGGGGG
Design oligonucleotides complementary to each Polymorphism.
These oligos are arrayed on a slide
Each spot corresponds to a polymorphism
Isolate genomic DNA from individual
Label Genomic DNA and hybridize to array
Oligo probes on slide
GACTCCTGAGGAGAAGTG
SNP1
GACTCCTGTGGAGAAGTG
GGGGGGGGGGGGGGGGGG
GGGGGGGGCGGGGGGGGG
SNP2
52
Take Genomic DNA from individual
Fragment DNA
Label DNA with fluorescent dye
53
Microarray
slide
GACTCCTGAGGAGAAGTG
SNP1
1
2
3
4
5
6
7
8
9
GACTCCTGTGGAGAAGTG
GGGGGGGGGGGGGGGGGG
SNP2
GGGGGGGGCGGGGGGGGG
Individua11
GACTCCTGAGGAGAAGTG
SNP1
GACTCCTGTGGAGAAGTG
1TT
2GG
GGGGGGGGGGGGGGGGGG
SNP2
GGGGGGGGCGGGGGGGGG
Individua12
GACTCCTGAGGAGAAGTG
SNP1
GACTCCTGTGGAGAAGTG
2CC
1AA
GGGGGGGGGGGGGGGGGG
SNP2
GGGGGGGGCGGGGGGGGG
Individua13
GACTCCTGAGGAGAAGTG
SNP1
GACTCCTGTGGAGAAGTG
1AT
2GG
GGGGGGGGGGGGGGGGGG
SNP2
GGGGGGGGCGGGGGGGGG
54
Genotype and Haplotype
In the most basic sense, a haplotype is a “haploid genotype”.
Haplotype: particular pattern of SNPs (or alleles) found on a
single chromosome in a single individual.
The DNA sequence of any two people is 99 percent identical.
Sets of nearby SNPs on the same chromosome are inherited in
blocks.
Therefore while Blocks may contain a large number of SNPs, a
few SNPs are enough to uniquely identify the haplotype of that
block.
The HapMap is a map of these specific SNPs.
SNPs that identify the haplotypes are called tag SNPs.
This makes genome scan approaches to finding regions with genes
that affect diseases much more efficient and comprehensive.
Haplotyping: involves grouping individuals by haplotypes, or
particular patterns of sequential SNPs, on a single chromosome.
There are thought to be a small number of haplotype patterns for
each chromosome.
Microarrays or PCR are used to accomplish haplotyping.
Haplotype and SNPs
Each individual has a characteristic pattern of SNPs
SNPs occur every 300-1000bp apart. There are over a million
SNPs in each individual
When we generate a SNP map for an individual we DO NOT
check every single SNP in that individuals DNA
SNPs are transmitted as blocks (Recombination hot spot)- so
no point analyzing SNPs that go together
1
aAa
2
cCc
3
A
4
G
5
G
6
T
7
A
8
T
9
C
Ind1
aGa
cGc
A
T
G
T
G
T
G
Ind2
SNPs in red were not studied. Only the 9 black SNPs were
studied
SNP mapping is used to narrow down the known physical location
of mutations to a single gene.
The human genome sequence provided us with the list of many of the
parts to make a human.
The HapMap provides us with indicators which we can focus on in
looking for genes involved in common disease.
By using HapMap data to compare the SNP patterns of people
affected by a disease with those of unaffected people, researchers
can survey the whole genome and identify genetic contributions to
common diseases more efficiently than has been possible without
this genome-wide map of variation: the HapMap Project has
simplified the search for gene variants.
57
A recessive disease pedigree
58
Mapping recessive disease genes with DNA markers
SNP markers are mapped evenly across the genome.
The markers are polymorphic.
We can tell looking at the SNP pattern of a particular
grandchild which grandparent contributed a certain part of its
DNA.
If we knew that grandparent carried the disease, we could say
that part of the DNA might be responsible for the disease.
1
2
3
4
5
4 different alleles at each locus
Position1 can be A or C or G or T
6
7
8
9
SNPs in red were
not studied. Only
the 9 black SNPs
were studied
Position2 can be A or C or G or T
Position3 ………………..
Grand
parent
Adam
A-A-A-A-A-A-A-A-A
Chromosome A-A-A-A-A-A-A-A-A
Carla
C-C-C-C-C-C-C-C-C
C-C-C-C-C-C-C-C-C
Gary
G-G-G-G-G-G-G-G-G
G-G-G-G-G-G-G-G-G
Tracy
T-T-T-T-T-T-T-T-T
T-T-T-T-T-T-T-T-T
59
Mapping recessive disease genes with SNP markers
1
Grand-parent
A
A-A-A-A-A-A-A-A-A
A-A-A-A-A-A-A-A-A
Dad
2 3 4
C
C-C-C-C-C-C-C-C-C
C-C-C-C-C-C-C-C-C
A-A-A-A-A-A-A-A-A
C-C-C-C-C-C-C-C-C
Offspring1
A-A-A-C-C-A-A-C-C
G-G-G-G-T-T-T-T-G
Offspring2
C-C-A-A-C-A-C-A-A
G-G-G-G-T-T-T-G-G
Offspring3
A-A-A-A-A-C-C-C-C
T-T-G-G-G-G-T-T-T
Offspring4
C-C-C-C-C-C-A-A-A
G-G-T-T-T-T-T-T-T
5 6
7
G
G-G-G-G-G-G-G-G-G
G-G-G-G-G-G-G-G-G
8 9
T
T-T-T-T-T-T-T-T-T
T-T-T-T-T-T-T-T-T
Mom G-G-G-G-G-G-G-G-G
T-T-T-T-T-T-T-T-T
Grandparents A and T and offspring 1 and 4 have the disease
We would look at the markers and see that ONLY at position 7
do offspring 1 and 4 have the DNA from grandparents A and T.
60
It is therefore likely that the disease gene will be somewhere
near marker 7.