Download Prof. Kamakaka`s Lecture 13 Notes

Basic techniques
--- Nucleic acid hybridization
complementary strands will associate and form double
stranded molecules
--- Restriction Enzymes
These enzymes recognize and cleave DNA at specific
sequences
--- Blotting
Allows analysis of a single sequence in a mixture
--- DNA cloning
This allows the isolation and generation of a large number
of copies of a given DNA sequence
--- Transformation
Stably integrating a piece of DNA into the genome of an
organism
--- DNA sequencing
Determining the array of nucleotides in a DNA molecule
--- PCR
amplification of known sequence
--- Genetic engineering
Altering the DNA sequence of a given piece of DNA
--- Genomics
Analyzing changes in an entire genome
1
Nucleic acid hybridization
Complementary strands of DNA or RNA will specifically associate
DNA is heated to 100C, the hydrogen bonds linking the two
strands are broken
The double helix dissociates into single strands.
As the solution is allowed to cool, strands with complementary
sequences readily re-form double helixes.
This is called Nucleic acid hybridization.
5’ AAAAAAAATTTTAAAAAAA 3’
Will associate with
3’ TTTTTTTTAAAATTTTTTT 5’
This occurs with complementary
DNA/DNA, DNA/RNA, RNA/RNA
2
Li-Fraumeni syndrome
This technique is very sensitive and specific.
A single 200 nucleotide sequence when added to a solution
of a million sequences will specifically hybridize with the
ONE complementary sequence
Usefulness
Li-Fraumeni syndrome
Individuals in a family have a propensity to develop tumors
at an early age
Often these families have a deletion in the p53 gene
When this family has a child, they might want to know
if their child has normal p53 or not
Nucleic acid hybridization provides a means to rapidly
determine whether the sequence is present or not
3
Sequencing
Genomic DNA
Fragment DNA
(clone)
Sequence fragments
ACGCGATTCA
ACGCGATTCA
Align fragments
GCGATTCAGGTTA
GATTCAGGTTA
CAGGTTACCACGC
ACGCGTAGCGC
TAGCGCA
TAGCGCATTACAC
ACGCGATTCAGGTTACCACGCGTAGCGCATTACAC Build consensus sequence
4
Sequencing
Reference Genome- Number of donor DNAs are sequenced
Pieces of DNA are sequenced many times
Computers are used to overlap the pieces to generate contigs
Consensus sequence is reference genome
Sequences of individuals will vary from the reference genome
ACGCGATTCAGGTTACCACGCGTAGCGCATTACAC
Reference Genome
ACGCGATTCAGGTTACCACGCGTAGCGCATTACAC
MISTY
ACGCGGTTCAGGTTACCACGCGTAGCGCATTACAC
NICK
ACGCGATTCAGGTTACCACGCGTAAAACATTACAC
JESSE
ACGCGGTTCAGGTTACCCCGCGTAGCGCATTACAC
DONNA
The sequence homology between Individuals is not perfect!!!
This allows us to assign a specific sequence to a specific
Individual
5
Homology (molecular biology)
Regions of the DNA (gene or non-gene) that share similar
nucleotide sequence
Sequence homology is a very important concept
Structural homology (nucleotide sequence) implies
functional homology
Genes with a similar sequence are likely to function
in a similar manner
Variation in sequence between individuals is also very
Important
6
The method
Isolate DNA
normal individual
Patient
Fragment DNA, Heat to denature
Add radiolabeled ssDNA (p53 gene)
(p53 probe)
Gradually and slowly cool solution
Radiolabeled p53 probe associates
with DNA in normal individual
If patient is deficient for p53 gene
Radiolabeled p53 probe is unable to
associates with DNA in patient
Add enzyme (nuclease) that specifically degrades
ssDNA molecules. dsDNA remains degraded
Radiolabel present in dsDNA
No radiolabel present in dsDNA
(because p53 probe could not anneal)
7
Restriction Enzymes
What are Restriction enzymes
What are restriction enzyme sites in DNA
How do we map Restriction enzyme sites in DNA
How do we use restriction enzymes to clone pieces of DNA
How do we use restriction enzyme sites/maps to study individuals
8
Restriction Enzymes
Enzymes which Recognize a SPECIFIC DNA sequence
BIND that sequence and
CUT The DNA at that specific sequence
SmaI is a Restriction enzyme
|
5’ AAAACCCGGGAAAA3’
3’ TTTTGGGCCCTTTT5’
|
This sequence is symmetrical. If one rotates it about the axis
It reads the same
EcoRI is another Restriction enzyme
|
5’ AAAAGAATTCAAAA3’
3’ TTTTCTTAAGTTTT5’
|
Some restriction enzymes recognize a specific sequence that is
4 bp long
Some restriction enzymes recognize a specific sequence that is
6 bp long
Some restriction enzymes recognize a specific sequence that is
8 bp long
9
BamHI
Restriction enzyme digestion of DNA (linear genomic
double stranded DNA)
OR
Restriction enzyme digestion of bacterial plasmid
DNA (small double stranded circular DNA)
No digestion of RNA
No digestion of single stranded DNA
Restriction enzymes
Linear/Circular DNA
No digestion of RNA
No digestion of single stranded DNA
A linear DNA molecule with ONE SmaI site will be cut into
two fragments
A circular DNA molecule with ONE SmaI site will generate
one DNA fragment
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12
Blunt Vs Sticky
Blunt ends
Sticky ends
After digestion of DNA by a restriction enzyme the DNA
ends are either blunt or sticky
Restriction sites
SmaI- BLUNT ENDS
5’AAAAAAAAAAGGGGGGGGTTTTTTTCCCGGGAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTTCCCCCCCCAAAAAAAGGGCCCTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGGGGGTTTTTTTCCC
3’TTTTTTTTTTCCCCCCCCAAAAAAAGGG
GGGAAAAAAAAGGGGGGGGTTTTTT3’
CCCTTTTTTTTCCCCCCCCAAAAAA5’
EcoRI is another commonly used restriction enzyme
5’AAAAAAAAAAGGGGGGGGTTTTTTTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGGGGGTTTTTTTG
3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAA
AATTCAAAAAAAAGGGGGGGGTTTTTT3’
GTTTTTTTTCCCCCCCCAAAAAA5’
Unlike SmaI which produces a blunt end,
EcoRI produces sticky or cohesive ends (SINGLE STRANDED)
These cohesive ends facilitate formation of recombinant
DNA molecules
14
5’AAAAAAAAAAGGGGGGGGTTTTTTTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGGGGGTTTTTTTG
AATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAA
GTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGTTTTTTTG
AATTCACGTACGTACGTACGTACGTACGTG
AATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTACCCCAAAAAAACTTAA
GTGCATGCATGCATGCATGCATGCACTTAA
GTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGTTTTTTTGAATTCACGTACGTACGTACGTACGTACGTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTACCCCAAAAAAACTTAAGTGCATGCATGCATGCATGCATGCACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’
15
Complementary sticky ends
AAAAAAGGGGGGGGTTTTTTTG
TTTTTTCCCCCCCCAAAAAAACTTAA
AATTCAAAAAAAAGGGGGGGGTTT3’
GTTTTTTTTCCCCCCCCAAA5’
AAAAAAGGGGGGGGTTTTTTTG AATTCAAAAAAAAGGGGGGGGTTT3’
TTTTTTCCCCCCCCAAAAAAACTTAA GTTTTTTTTCCCCCCCCAAA5’
GGCCCAAAAAAAAGGGGGGGGTTT3’
AAAAAAGGGGGGGGTTTTTTTG
GTTTTTTTTCCCCCCCCAAA5’
TTTTTTCCCCCCCCAAAAAAACTTAA
Enzyme compatibility
SmaI
AAACCCGGGAAA
TTTGGGCCCTTT
EcoRI
KpnI
AAAGAATTCAAA
TTTCTTAAGTTT
AAAGGTACCAAA
TTTCCATGGTTT
XmaI
MfeI
AAACCCGGGAAA
TTTGGGCCCTTT
AAACAATTGAAA
TTTGTTAACTTT
Asp718 AAAGGTACCAAA
TTTCCATGGTTT
xxxxxxx
18
Restriction maps
Restriction maps are descriptions of the number, type and
distances between Restriction sites on a piece of DNA.
Very useful for molecular biologists.
Previously we used specific genes as markers on chromosome and
Map units to indicate distance between the markers.
Its like using specific landmarks to identify your location along
a road. Restriction sites are also used as landmarks along a
piece of DNA.
pr
11Mu
vg
5Mu
cy
300kb
PstI
HindIII
EcoRI
205kb
4kb
SmaI
Vg
Pr
NNNNNNNNNGAATTCNNNNNNNNNNNNAAGCTTNNNNNNNNNNNNCTGCAGNNNNNNNNNNCCCGGGNNNNNN
NNNNNNNNNCTTAAGNNNNNNNNNNNNTTCGAANNNNNNNNNNNNGACGTCNNNNNNNNNNGGGCCCNNNNNN
AAAAAAAAAGAATTCTTTTTTTTTTTTAAGCTTCCCCCCCCCCCCCTGCAGGGGGGGGGGGCCCGGGAAAAAA
TTTTTTTTTCTTAAGAAAAAAAAAAAATTCGAAGGGGGGGGGGGGGACGTCCCCCCCCCCCGGGCCCTTTTTT
Restriction sites CAN serve as MARKERS ALONG the DNA.
They can be used to generate a physical map of a specific DNA
sequence can be created.
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Restriction maps
Human Genome is 1.5 billion basepairs long
There are 25,000 genes (markers)
Every gene is on average approximately 600,000 bp apart
EcoRI sites are on average 4000 bp apart
HinDIII sites are on average 4000 bp apart
Etc etc
There are many more Restriction enzyme sites (landmarks) on
any one piece of DNA then there are genes (landmarks)
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Sequence Divergence
The restriction map is a partial picture of the nucleotide sequence
of a gene.
By comparing restriction maps we can surmise differences in the
sequence between species
NNNNNNNNGAATTCNNNNNNNNNNNNNNNNAAGCTTNNNNNNNNNNNNNNCTGCAGNNNNNNNNNNNNNNN
NNNNNNNNCTTAAGNNNNNNNNNNNNNNNNTTCGAANNNNNNNNNNNNNNGACGTCNNNNNNNNNNNNNNN
GeneA
Mai California me rahta hu aur UCSC me padhata hu.
Human
Chimp
Gibbon
21
Mai California me rahta hu aur UCSC and UCLA me padhata
hu.
Mai California me rahta hu aur mai sirf
UCLA me padhta hu
Deletions and additions
Normal Globin gene
HindIII
EcoRI
4
Globin gene from a thallesimia
patient
4
HindIII
3
EcoRI
5
HindIII
EcoRI
EcoRI
3
8
HindIII
5
EcoRI
EcoRI
3
With restriction maps, the relationship between a gene from
two different individuals can be determined without having to
actually sequence the gene from both individuals.
22
Very Large deletions or insertions can be studied using
microscopy
Small to large deletions/insertions (100 bp to several kb) can be
studied using restriction maps!!
23
Describing a DNA piece based on the pattern of restriction sites
Restriction map of a cloned piece of DNA
Linear or circular
B
K
Restriction map
S
B
E
H
E
B
K
S
B
How do I do this for an unknown piece of DNA?
Analogy
1
2
a
c
Goal: Identify the
parts for this blob
and describe the
spatial relationship
between the parts
Break the blob apart into
its separate parts
Look at the shapes and
numbers of the parts
b
3
a
c
b
Fit the parts together
Restriction map of piece of DNA
Large amount of pure DNA (many copies of the same DNA is
required) (Cloned)
Take (cloned) DNA in a tube,
Add restriction enzyme,
Allow enzyme to cut DNA at its binding sites
Run the digested DNA on a gel to resolve the DNA
fragments based on size
Piece the fragments together to determine the linear order
of the fragments (build the map)
Method
-
DNA is an anion (-ve charged)
and moves towards the
Positive anode.
7
The rate of migration of a
DNA fragment is inversely
proportional to its size.
Larger the size, slower its
movement.
3
4
2
+
1
2
HindIII
5
EcoRI
3
HindIII
EcoRI
EcoRI
1
5
27
EcoRI/HindIII
The length of the DNA can
be accurately determined by
allowing the charged DNA to
run through an agarose gel.
HindIII
Agarose gel electrophoresis
Marker
EcoRI
Marker
EcoRI
HindIII
EcoRI/HindIII
Gel electrophoresis
Mapping
20
14
HindIII
Marker
uncut
EcoRI
You are given a 20 kb fragment of DNA
After trying many enzymes you find that EcoRI and
HindIII cut the fragment
HindIII 14kb and 6kb
EcoRI 12kb 6kb and 2kb
Solve the map
6
H
14
12
6
4
2
1
6
14
Or its mirror image
14
6
They are the same
What about EcoRI?
12
2
E
6
E
28
How do you arrange these three fragments wrt one another
Mapping
Since HindIII cut the 20kb fragment once, in which of the
three EcoRI fragment does it cut?
Marker
EcoRI
HindIII
EcoRI+HindIII
A double digest with both enzymes will provide the answer
Fragments of 8kb, 6kb, 4kb and 2kb
The double digest does not alter the
size of the 6kb and 2kb fragments
The 12kb fragment is lost. Also 8+4=12
14
12
6
4
8
4
2
6
12
2
1
8
H
4
E
6
E
Partial Map only!!!! Not enough information
29
2
New Mapping
How are these fragments ordered?
Marker
EcoRI
HindIII
EcoRI+ HindIII
The HindIII single digest tells us that they must be ordered so
that One side adds up to 6kb and the other side adds up to 14kb
14
12
6
4
2
1
30
Mapping
HindIII
14
6
EcoRI
12
6
2
HindIII/EcoRI
8
6
4
2
31
Mapping
HindIII
14
6
EcoRI
12
6
HindIII/EcoRI
8
6
4
2
2
4
6
2
4
2
6
8
6
4
16 & 4
12
4
8
6
2
12
4
8
6
12 & 8
12
8
2
16 & 4
12
4
2
12 & 8
12
8
6
HindIII
8
12
10 & 10
14 & 6
2
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EcoRI+ PstI
PstI
EcoRI+ HindIII
EcoRI
HindIII
Marker
Another linear map
14
12
8
6
4
2
1
33
Different Mapping example
Hi
12
8
Ec
12
6
2
Hi/Ec
8
6
4
2
Ps
13
7
Ps/Ec
12
5
2
1
Three different enzymes
Hi
Ec
Ps
34
Mapping
HindIII
12
8
EcoRI
12
6
2
HindIII/EcoRI
8
6
4
2
HindIII
12 & 8
12 & 8
35
Mapping
HindIII
12
8
EcoRI
12
6
HindIII/EcoRI
8
6
4
2
HindIII
2
16
8
6
E
2
E
4
E
2
E
12
2
E
6
H
16 & 4
8
12 & 8
12
4 H
E
4
12
12
6
H
8
12 & 8
12
36
Mapping
EcoRI
12
6
2
PstI
13
7
PstI/EcoRI
12
5
2
1
37
Mapping
EcoRI
12
6
2
5
P
1
P
6
1
12
E
E
1 & 19
E
E
2
6
P
12
E
12
E
5
6
5
2
2
5 & 15
5
2 E
PstI/EcoRI
12
5
2
1
Pst I
1
E
6
2
PstI
13
7
6
3 & 17
12
E
P1
7 & 13
12
E
4
8
H
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Final Map
P
5
2
Hi
12
8
1
8
4
E
E
Ec
12
6
2
H
Hi/Ec
8
6
4
2
Ps
13
7
Ps/Ec
12
5
2
1
39
Mapping deletions
Say you isolated this DNA from a region coding for GeneA, from
a normal Patient and one suffering from a syndrome.
The fragment was 17kb in the affected individual rather than
20kb in the normal patient
You draws restriction maps for the normal patient
6
E
8
H
4
E
2
You draws restriction maps for the affected individual
6
E
8
H1 E
2
There is a 3kb deletion in the 4kb HindIII/EcoRI fragment
40
Complex Mapping
Marker
2kb
2kb+HindIII
Marker
6kb
6kb+HindIII
Marker
EcoRI
HindIII
EcoRI+
HindIII
Marker
12kb
12kb+HindIII
Often maps are more complex and difficult to analyze using
single and double digests alone.
To simplify the analyses, you can isolate each EcoRI band
from the gel and then digest with HindIII
14
14
14
14
12
12
12
12
6
6
6
6
4
4
4
4
2
2
2
2
1
1
1
1
41
Mt DNA
Y chromosomes can be used to study paternal lineage
mtDNA can be used to follow maternal lineage
Cells contain organelles- Mitochondria are organelles that produce
Energy. They contain a small 17,000 bp circular DNA.
It encodes for 13 proteins in human cells and some tRNA’s
Hypervariable region (150 bp)
tRNA
CytochromeB
NADH dehydrogenase
cytochromeC oxidase
ATP synthase
Mitochondrial DNA inheritance is not mendelian
It is inherited maternally
42
Using DNA to study History
This hypothesis was initially derived from restriction maps of
mitochondrial DNA
Australia
Europe
Asia
Africa
“Eve’s DNA”
43
All humans are derived from a small African population about
170K yrs ago
Eve
Geographic region
DNA
A
Mutation generates B from A. Now you have individuals
With A and B DNA in population.
C
B
A
B
A
D
44
Out of Africa
C
C
B
C
F
A
D
E
B
A
D
G
C migrates to form a separate population.
Additional mutations diversify DNAs in populations.
Original population more diverse than newer population
Compared sequences of mtDNA
There are greater sequence differences among Africans than
any other group (Europeans, American Indians, Asians, etc etc)
The african population had the longest time to evolve variation
And thus humans originated in Africa.
45
Xxxxxx
46
THE GENE PROBE!!!
How do we isolate a GENE?
How did we get a pure copy of the gene?
47
Cloning DNA
A reasonable question is how did we clone a fragment of DNA
Or how do we clone a gene
The construction of Recombinant DNA molecules or cloning of
DNA molecules
Recombinant DNA is generated through cutting and pasting of
DNA to produce novel sequence arrangements
Restriction enzymes such as EcoRI produce staggered cuts
leaving short single-stranded tails at the ends of the
fragment.
These “cohesive or sticky” ends allow joining of different
DNA fragments
When a piece of DNA is cut with EcoRI,
you get
nnnG
nnnCTTAA
|
nnnGAATTCnnn
nnnCTTAAGnnn
|
AATTCnnn
Gnnn
48
Cloning DNA
A reasonable question is how did we get the 20kb fragment of
DNA in the first place?
To understand the origin of the fragment we must address the
issue of:
The construction of Recombinant DNA molecules or cloning of
DNA molecules
Recombinant DNA is generated through cutting and pasting of
DNA to produce novel sequence arrangements
49
Recombinant DNA
Restriction enzymes such as EcoRI produce staggered cuts
leaving short single-stranded tails at the ends of the fragment.
These “cohesive or sticky” ends allow joining of different DNA
fragments
When a piece of DNA is cut with EcoRI,
you get
|
GAATTC
CTTAAG
|
AATTC-----------------G
G-----------------CTTAA
AATTC-----------------G
G-----------------CTTAA
5’AAAAAAAAAAGGGGTTTTTTTG
AATTCAAAAAAAAAAAAAAGGGGGGGGTTTTTTTG
AATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTACCCCAAAAAAACTTAA
GTTTTTTTTTTTTTTCCCCCCCCAAAAAAACTTAA
GTTTTTTTTCCCCCCCCAAAAAA5’
50
Plasmids
Plasmids are naturally occurring circular pieces of DNA in E. coli
The plasmid DNA is circular and usually has one EcoRI site.
It is cut with EcoRI to give a linear plasmid DNA molecule
AATT
51
Plasmids
Small circular autonomously replicating extrachromosomal DNA
Modified plasmids, called cloning vectors
are used by molecular biologists to isolate
large quantities of a given DNA fragment
Plasmids used for cloning share three
properties
Unique restriction site
Antibiotic resistance
Origin of replication
E B
Bacterial genome Plasmid DNA
(5000kb)
(3kb)
Origin
52
Antibiotic
resistance gene
Plasmid elements
Origin of replication:
This is a DNA element that allows the plasmid to be replicated
and duplicated in bacteria.
Each time the bacterium divides, the plasmid also needs to divide
and go with the daughter cells. If a plasmid cannot replicate
in bacteria, then it will be lost.
53
Plasmid elements
Antibiotic resistance:
This allows for the presence of the plasmid to be selectively
maintained in a given strain of bacteria
+antibiotics
-antibiotics
Lab bacterial strains are sensitive to antibiotics.
When grown on plates with antibiotics, they die.
The presence of a plasmid with the antibiotics resistance gene
allows these lab strains to grow on plates with the antibiotic. You
are therefore selecting for bacterial colonies with the Plasmid
54
Plasmid elements
Unique restriction sites:
For cloning the plasmid needs too be linearized. Most cloning
vectors have unique restriction sites. If the plasmid contains
more than one site for a given restriction enzyme, this results
in fragmentation of the plasmid
Why does this matter?
Ori
Antibiotic
resistance
gene
55
pUC18
pUC18 is one of the most commonly used plasmid:
pUC= plasmid University of California
Plasmid
pBR322
pUC18
pACYC
pSC101
replicon
pMB1
pMB1
p15A
pSC101
copy No
15
500
10
5
56
Cloning DNA
When a piece of DNA is cut with EcoRI,
you get
|
GAATTC
CTTAAG
|
AATTC-----------------G
G-----------------CTTAA
AATTC-----------------G
G-----------------CTTAA
When tow pieces of DNA cut with EcoRI are ligated back
together you get back an EcoRI site
-----------------G AATTC---------------------------------CTTAA G-----------------
57
58
Ligation
PLASMID
GENOMIC DNA
AATT
The EcoRI linearized PLASMID DNA is mixed with HUMAN
DNA digested with EcoRI
The sticky ends will hybridize/anneal specifically and a
recombinant plasmid will be generated
59
Cloning
The genomic DNA fragments is mixed with a plasmid that has
been linearized at a single EcoRI site (say pUC18)
E
F
G
h
Ampr
d
Ori
Both the plasmid and genomic DNA have been cut with EcoRI,
they have complementary sticky ends
|
G A A T T C
C T T A A G
|
________________________ AATT----------------------------- AATT
________________________TTAA -----------------------------TTAA
Plasmid
Genomic DNA
60
Recombinant plasmid
This process where foreign genomic DNA is joined to plasmid
DNA is called ligation
It results in recombinant plasmid (foreign DNA+plasmid)
Each plasmid has one foreign EcoRI fragment
Each foreign fragment is still present as only one copy! This is
not useful.
61
Incompatibility of sticky ends
|
Plasmid cut with EcoRI
G A A T T C
C T T A A G
|
_____________________
_____________________TTAA
AATT
Plasmid
Genomic DNA cut with HinDIII
|
A A G C T T
T T C G A A
|
AGCT-----------------------------------------------------TCGA
Genomic DNA
Won’t work!!
__________________ AGCT----------------------------AATT
__________________TTAA -----------------------------TCGA
62
Transformation
Ampr
The entire collection of these plasmids bearing genomic DNA
inserts is called a Genomic Library!
These plasmids are added back into bacteria by a process
called transformation
Ori
The bacteria are selected for the presence of the Plasmid by
growth on media containing antibiotics
dE F
G h
Petri dish + antibiotic
Each colony of E. coli will harbor one plasmid with one piece of
genomic DNA. Only cells with plasmid will grow on plates with
antibiotics (the antibiotic resistance gene on plasmid allows these
63
cells to grow). Cells that did not take up a plasmid will not grow.
Plasmid propagation
The plasmid DNA can replicate in bacteria
and therefore many copies of the plasmid
will be made. The human DNA fragment in
the plasmid will also multiply along with
the plasmid DNA.
THE DNA IS CLONED
Normally a gene is present as 2 copies in
a cell. If the gene is 3000bp long there
are 6x103 bp in a total of 6x109 bp of
the human genome
Once ligated into a plasmid, unlimited
copies of a single gene can be
produced.The process of amplifying and
isolating the human DNA fragment is
called DNA cloning.
64
Why are plasmids important?
Most genes are present as two copies in the entire genome.
Plasmids allow us to obtain 1000’s of copies of a gene in a pure
form
65
Cloning genes
Cloning and Expression of genes in cells
Coding region of protein
Enhancer, Promoter, Ribosome binding site
E B
No cloning of RNA into
double stranded plasmid
DNA
No cloning of single
stranded DNA into double
stranded plasmid DNA
E B
E
E
H
Coding region cloning
E
H
E
B
B
E
H
E
B
B
K
S
B
Promoter cloning
E
H
E
B
K
S
B
Isolate the plasmid
To isolate the gene fragment, we grow up a large population of
E. coli containing the plasmid with the gene insert.
A simple procedure allows us to isolate the plasmid (which is
smaller than Chromosomal DNA)
Once we have purified the plasmid we have 1000’s of copies of
Gene in a plasmid
We can take the plasmid and cut it with EcoRI. When the
digest is run on an agarose gel, we get two bands- one
corresponding to the plasmid and one to the insert.
EcoRI
Uncut
Marker
The DNA present in the band corresponding to the insert can
be isolated from the gel
PURE GENE !!!!!
Gene C
plasmid
69
Foreign gene expression
What if you want to express Influenza antigen in chicken cells?
Influenza virus promoter sequences do not work in chicken cells
Connect Influenza antigen gene to a chicken enhancer/promoter
Chicken
Enhancer
Chicken
Promoter
Influenza Gene
70
Mixing and matching
HinD
Blood specific
promoter
5’UT
R
HinD
Coding region GLOBIN
gene
3’UT
R
ori
Kanr
HinD
Liver specific promoter
ori
Kanr
HinD
HinD
Globin Expression in liver
71
Inter-species Gene transfer
CF gene on a
plasmid
CF+
Isolate Plasmid
Transfect human cell
with CF+ plasmid
Human Cell is cf-/cfIt becomes CF+ after transfection
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Definition of Key Terms
Cisgenics
Genetic modification of a recipient plant/animal with a gene
from a sexually compatible plant/animal species
Transgenics
Genetic modification of a recipient plant with a gene from a
sexually incompatible plant/animal or other organism
Traditional breeding
Conventional cross breeding of two species of plants to
transfer a gene from one species to the other
Are cisgenics acceptable?
Are transgenics acceptable?
73
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Genetically modified organisms GMO
Attempts to cross wheat and rye produce sterile
offspring.
New techniques were developed that allowed production
of fertile hybrid.
The two plants were treated with a potent toxin
colchicine and the genomes were severely
MUTAGENIZED
The mutagenesis allowed the genome of wheat and rye
(these are different species) to overcome the species
barriers, fuse and form a NEW SPECIES !!
These plants were used to develop genetically novel
plants with traits from wheat and rye parents
producing a “SuperFood”
GOOD IDEA?
BAD IDEA?
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Inactivating single genes
Ethylene gas released by fruit accelerates the ripening
process. Prevention of ethylene production would block the
fruit from ripening prematurely and spoiling on the way to the
market.
The ethylene biosynthetic pathway is as follows:
Precursor----->ACC------>ethylene
ACC
ACC
synthase
oxidase
Technology was used to generate mutants in the plant so that they
could not synthesize the enzymes required for ethylene gas
production.
Generated and marketed the “SUPER SAVR TOMATO”
GOOD IDEA?
BAD IDEA?
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Expressing a foreign gene
A species of bacteria produces a potent natural pesticide
This pesticide is used in organic farming
The gene necessary for producing the toxin was identified and
cloned.
The gene was inserted into the genome of plants.
This bacterial gene was now able to replicate in plants and the
plant made and secreted the toxin. The plant now produced the
toxin thus eliminating the need for pesticide spraying.
This reduces the harmful effects of pesticides on humans
However, insects start becoming resistant to this toxin.
GOOD IDEA?
BAD IDEA?
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Reintroducing WT gene
Ancient native corn (roots) emit a volatile substance, bcaryophyllene, when attacked by insects. The substance
attracts nematodes to the roots. These worms eat the insects
protecting the corn.
Commercial corn has a mutation and cannot produce bcaryophyllene.
The wild type gene b-caryophyllene synthase was cloned.
A commercial corn plant was transformed with the wild type
gene -b-caryophyllene synthase. The plant could now produce
b-caryophyllene and was resistant to insects.
Good Idea
Bad Idea?
Ancient corn
Small cob
Large height
Insect resistant
Slow growth
Easily stressed
x
commercial corn
large cob
short height
insect sensitive
rapid growth
stress resistant
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Is the trait present in close relatives
Yes
Conventional breeding or Cisgenics
Parent1
x
(start with ~1000 crosses)
Parent2
F1
Phenotype selection
(500,000 plants)
NO
Transgenics
(insert gene from other
organisms)
F2
(50,000 plants)
F3
(asses using markers)
F4
(5000 plants)
F5
(1000 plants- check yields, other traits)
F6
(5 plants- submit for official trials)
(Linked genes along with trait desired)
or Cisgenics
Insert single gene lost from
ancestor
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Genetically modified plants
Trititcale- created in the 1880’s-1930’s by the Edinburgh Botanical
Society. Using chemical mutagenesis combined with Mendelian
crosses. It is currently sold primarily in organic health food stores.
Is it a good idea to mutate crops using chemical mutagens?
Flavor Savr tomato helps transport fragile food preventing waste.
Labeled a Frankenfood. It has a single mutation in one gene. Is it a
good idea to mutate crops using recombinant DNA methods? What
if you made the same mutation by classical genetics?
Bt cotton created in the 1990’s using recombinant DNA and
transgenic technology. What if you inserted a gene from one species
in to another species using classical genetics?
Reinserting Caryophyllene synthase into corn restores its natural
insect resistance which was lost when commercial corn varieties
were generated by classic breeding techniques. What if you inserted
this gene back by genetic crosses?
Gene blocking may produce tea, coffee without the caffeine
Tomatoes with a higher antioxidant (lycopene) content
Fungal resistant bananas
Smaller, seedless melons for use as single servings
Bananas and pineapples with delayed ripening qualities
http://www.nytimes.com/2013/03/19/science/earth/research-tobring-back-extinct-frog-points-to-new-path-andquandaries.html?pagewanted=all
Message:
Understand the differences
(GM-foods) pre- and post-1990
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81
Types of clones
What are genomic clones
What are cDNA clone
What is a PCR clone of a specific gene
Genomic clones
Genomic DNA
Digest with restriction enzyme
Ligate with plasmid
Grow individual plasmids in E. coli
Genomic DNA
Digest Genomic DNA and plasmid with restriction enzyme
Ligate with cut plasmid DNA
Grow individual plasmids in E. coli
A
Gene1
Gene2
B
C
Gene3
D
E
F
Each fragment is ligated into the plasmid
Each plasmid is put (transformed) into E.coli
Each E. coli colony on a plate has one specific plasmid
C
D
A
B
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Genomic clone libraries
Species
Genome size
average
insert size
#plasmids
E. Coli
Drosophila
Human
5000kb
150,000kb
3000,000kb
16 kb
16 kb
16 kb
1300
46,000
>100,000
An entire genome of any organism can be cloned as small
fragments in plasmids
The larger the genome, the more difficult the task
At present, genomic DNA libraries exist for a large number of
organisms including
Yeast, C.elegans, Drosophila, Zebrafish, Xenopus, Chickens,
Mouse, Humans etc
86
cDNA clone
RNA Cannot be cloned
Reverse transcriptase copies RNA into DNA
So to clone RNA, you first convert RNA into DNA using
reverse transcriptase
This DNA (cDNA) is an complementary copy of the RNA
(RNA was the template)
The cDNA is then cloned into plasmids
cDNA
Often we have RNA rather than DNA as the starting material
For instance in the case of the human hemoglobin gene, we
started with globin mRNA
RNA is difficult to work with. In contrast to DNA, RNA breaks
down and degrades very easily. There are no restriction enzymes
that cut RNA at specific sites.
RNA cannot be cloned. It cannot be inserted into a plasmid and
amplified since all Plasmids are DNA.
The enzyme reverse transcriptase has proven very useful to
molecular biologists.
This enzyme catalyzes the synthesis of DNA from a RNA
template. It is normally found in a large class of viruses. The
genome of these viruses is RNA!! These viruses are called
retroviruses.They infect eukaryotic cells and use these cells to
grow/replicate
Retroviruses carry an RNA genome. Interestingly they will
integrate into the DNA of the host. For RNA to integrate into
DNA, first the RNA has to be converted to DNA
Remember the central dogma of molecular biology
Information flows from DNA to RNA to protein!
DNA---->RNA---->protein
Reverse Transcriptase reverses this dogma (partially)
88
cDNA synthesis
Protein coat
RNA genome
Reverse
transcriptase
mRNA
mRNA
DNA
RT
DNA
cDNA
89
cDNA/splicing
So from globin mRNA, a complementary DNA molecule can be
created using reverse Transcriptase. This complementary DNA is
called cDNA.
The cDNA can now be inserted into a plasmid and cloned.
What is the relationship between a cDNA clone and a genomic
clone?
Splicing
In eukaryotes, the coding sequences are interrupted by introns
1 2
3
4 5
Gene
7700 nt
6
7
Ovalbumin
Primary
transcript
Splicing
mRNA
1872 nt
90
Genomic Vs cDNA
Genomic clones represent the organization of the DNA in the
nucleus!
cDNA clones represents the organization of mRNA sequences
after the gene has been transcribed, processed and exported to
the cytoplasm.
cDNA clones contain the sequence of nucleotides that code for
the mRNA--protein!
cDNA clones do not contain the sequence of the promoter of the
gene or the intron.
The starting material for cDNA clones is different from material
used to make genomic clones
Genomic clone
cDNA clone
Source
Nucleii
(any cell)
cytoplasmic RNA
(specific cell type)
Use
Studies on gene
organization &
Studies directed
towards coding
regions
structure
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PCR
It’s a method that can be used to make many copies of a
particular DNA sequence from a particular individual
You have to know the DNA sequence before you can amplify
that sequence (it does not have to be cloned)
The sequence will not propagate (replicate) in living
organisms
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PCR
Heat 95C to denature DNA
and add primers
Let Primers hybridize to DNA (55C)
Add Heat resistant DNA polymerase and dNTP (70C)
Repeat- 95C55C70C
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94
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
PRIMER1 5’AAAGATC3’
3’AGCTAGAT5’ PRIMER2
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’AGCTAGAT5’
5’AAAGATC3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCC AGCTAGAT5’
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
95
PCR
How do you detect PCR?
Agarose Gels
Size of PCR product will depend upon location of PCR primers
96
PCR clone
PCR cloning
IF YOU KNOW THE SEQUENCE OF THE GENE YOU
WANT TO CLONE
You can use PCR to first make many copies of your gene
Then you cut the PCR fragment and plasmid with a
restriction enzyme
Ligate PCR with plasmid, transform E.coli
Then you can clone those copies into a plasmid.
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Blotting
You can Restriction map a cloned piece of DNA
Can you restriction map a piece of DNA in the genome without
first cloning it?
99
Southern blotting
Rapid method of identifying a specific DNA fragment from a
mixture of fragments (or from different individuals)
plasmid
EcoRI
EcoRI
Uncut
Marker
Insert
Gene
How do you determine which
band corresponds to insert and
which to the plasmid
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Marker
Uncut
EcoRI
EcoRI
Uncut
Marker
A probe is used to identify genomic DNA?
DNA is
transferred from
the gel to a paper
filter
The DNA (plasmid and chromosomal) on the paper is denatured
(converted from ds to ss)
Then you take the filter and to it add radiolabeled probe (small
part of Gene).
101
Southern blotting with a probe
gatcgatcgatcTTTTTTTgatcgatc
AAAAAAA
Marker
Uncut
A probe with this specific sequence is
generated and made radioactive
EcoRI
The probe AAAAAAA will bind the single
stranded DNA that has a complementary
sequence (TTTTTT). It will specifically
hybridize with the insert (genomic DNA)
Incubate the filter with the radiolabeled probe
A specific probe enabled us to identify
a DNA fragment that corresponds to a
specific gene of interest.
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PROBES
Probes are obtained in a number of ways
RNA as a source
The probe for hemoglobin can be obtained from mRNA of
immature red blood cells.
The major transcript of these cells is from the hemoglobin
gene. So isolating RNA from these cells, we can obtain a
relatively pure probe for the hemoglobin gene
Protein
If you have a purified protein, the amino acid sequence can
be determined.
From the amino acid sequence, using the genetic code a
corresponding DNA sequence can be synthesized and this
small DNA piece can be used as a probe
Homology
Probes from conserved genes-Many genes are conserved from
one species to another
Chimpanzee and human DNA are 97% identical. If you know
the sequence of a gene in chimps, then you will be able to
know the sequence for the gene in humans!
The histone genes are highly conserved across phyla. Histone
proteins have three Amino acid differences between humans
and peas
Histone genes have been isolated in yeast, they can serve as
probes for screening a Human genomic library- cloning by
phone
The computer databases
PCR
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What about a genome?
What if Gene C was in a large genome. Could we identify the
fragment by Southern blotting
EcoRI
2kb
4kb
5kb
GeneC
GeneX
Marker
Marker
GeneR
3kb
Transfer to
membrane
4.5kb
0.5kb
GeneA
Marker
1kb
Hybridize with
Probe C
Based on the blot what is the restriction map for gene C?
104
Map by Blotting
Actual Map
E
1kb
E
2kb
E
E
3kb
4kb
GeneR
5kb
GeneC
GeneX
8kb
H
E
4.5kb
0.5kb
GeneA
9kb
Marker
H
Marker
3kb
E
EcoRI
Southern Blot inferred Map
E
E
4kb
GeneC
105
You can build a more detailed genomic restriction map
2kb
E
3kb
E
4kb
GeneR
5kb
GeneC
GeneX
8kb
H
H
Marker
Marker
3kb
E
EcoRI
E
4.5kb
0.5kb
GeneA
9kb
Marker
1kb
E
Marker
E
HindIII
Southern Blot inferred Map
E
E
4kb
GeneC
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H
8kb
H
You can build a more detailed genomic restriction map
Marker
Marker
Marker
If we digest the DNA with HindIII and EcoRI what will
happen?
HindIII
EcoRI
EcoRI+
HindIII
Southern Blot inferred Map
E
E
4kb
GeneC
H
8kb
H
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E
E
E
1kb
E
E
2kb
4kb
3kb
GeneC
ProbeA
ProbeB
ProbeC
Mapping chromosomal DNA with different probes
Probe A
2Kb fragment
Probe B
2Kb and 4Kb fragment
ProbeC
4Kb fragment
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E
E
1
Restriction mapping Individuals
E
4
E
2
Gene
A
E
1
E
1
E
B
E
4
Gene
A
WT
Mutant
WT
Marker
Marker
Marker
Marker
B
Mutant
Mapping deletion with probe A. Mapping deletion with probe B.
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110
Northern blot
This is a rapid method that allows you to determine the cell
type in which a specific gene is active and being
transcribed.
Brain
Bone
Embryo
Blood
Lung
liver
These tissues differ because each is transcribing a
unique subset of genes.
Each tissue contains a unique and distinct mRNA
population
Presence of RNA is a reflection of gene activity
111
Northern blot
Method is analogous to Southern blots
Instead of DNA as the starting material, you use RNA.
You take cells, break them open, isolate the RNA and run the
RNA on a gel
Transfer RNA to membrane and use probe for gene of
interest.
WT individual
Globin probe
Kidney
Bone marrow
Brain
erythrocyte
Lymphocyte
Kidney
Bone marrow
Brain
erythrocyte
Lymphocyte
The RNA can be from specific tissues or cell types
mutant individual
Globin probe
Presence of RNA is a reflection of gene activity
112
Microarray
Microarrays are miniature devices containing thousands of DNA sequences stuck on
at different positions (addresses). Hybridisation to complex mixtures of labelled
DNA molecules, prepared from cellular RNA, shows the relative expression levels of
thousands of genes. This can be used to compare gene expression levels within a
sample or look at differences in the expression of specific genes across different
samples.
Key principles
* RNA isolated from a particular cell type or tissue comprises a complex mixture
of different RNA transcripts. The abundances of individual transcripts in the
mixture reflect the expression levels of the corresponding genes.
* A microarray is a small device, about the size of a microscope slide, with
thousands of different known DNA sequences immobilised at different addresses on
the surface.
* Each of these DNA sequences can participate in a hybridisation reaction.
* If a complex DNA mixture copied from the above RNA is labelled and hybridised
to the microarray, the strength of the signal at each address shows the relative
expression levels of the corresponding gene.
Microarrays can be used to compare gene expression levels within a sample or look at
differences in the expression of specific genes across different samples.
How does it work?
Single DNA strands with complementary sequences can pair with each other and form
double-stranded molecules. This hybridisation process occurs with such specificity
that a labelled DNA probe - a single DNA strand carrying a label that allows it to be
detected — can pick out a matching partner, the target, in a complex mixture
containing millions of different sequences.
Microarrays apply the hybridisation principle in a highly parallel format. Instead of
one target, thousands of different potential targets are arrayed on a miniature solid
support. Instead of a unique labelled DNA probe, a complex mixture of labelled DNA
molecules is used, prepared from the RNA of a particular cell type or tissue.
The abundances of individual labelled DNA molecules in this complex probe reflect
the expression levels of the corresponding genes. When hybridised to the array,
abundant sequences will generate strong signals and rare sequences will generate
weak signals.
The strength of the signal thus represents the level of gene expression in the
113
original sample.
How is it used?
Microarrays
These are reverse northern blots.
Allows us to examine gene expression of all of the genes in the
genome!
Each spot is DNA for one defined gene.
Each gene DNA is spotted in a grid.
They cover the entire genome.
Make total RNA from normal and
mutant cell,
Label each total RNA differently
Wt=red
Mut=green
Add labeled RNA from normal
and mutant cells to array and let
hybridize
Measure label and determine
change
WT Mut
1
2
3
4
5
Ratio of WT/mut
114
1, 2, 3 … are sequences specific
for gene1, gene2, gene3 etc
printed on the slide
Use of microarrays
To measure changes in transcription of genes during drug
treatment
To identify deletions in DNA
A microarray works by exploiting the ability of a given
mRNA molecule to bind specifically to, or hybridize to,
the DNA template from which it originated.
115
Genome sequencing
Whether bacterium or human, the genome of any organism to too large to be deciphered in one go.
The genome is therefore broken into smaller pieces of DNA, each piece is sequenced and computers
fit all the sequences back together.
The human chromosome to be sequenced.
The chromosome is first chopped randomly into conveniently sized chunks.
These large fragments are inserted into bacterial artificial chromosomes (BACs) and cloned in
bacteria.
These fragments are then mapped so it is known which region of the chromosome they came from.
Each BAC is shotgunned - broken randomly into many small pieces. This process is repeated several
times to give different sets of fragments. (The whole-genome shotgun method goes directly to this
stage.)
The fragments are cloned in small vectors and then sequenced. About 500 bases of sequence
information is produced from each fragment.
The sequences are fed into a computer, which looks for overlaps at the end of the sequence to find
neighbouring fragments.
When many fragments have been sequenced the sequence of the original BAC insert can be
assembled. The process is carried out for all the BACs to give a complete chromosomal sequence.
For example, the human genome is about 3 billion base pairs, arrayed in 24 chromosomes. The
chromosomes themselves are 50–250 million bases (megabases) long. These tracts of DNA are much
too large for even the latest automated machines, which sequence fragments of DNA between 400
and 700 bases long.
The genome is first broken into conveniently sized chunks, fragments of about 150 kilobases. Each
fragment is inserted into a bacterial artificial chromosome (BAC), a cloning vector used to propagate
DNA in bacteria grown in culture.
The BACs are then mapped, so that it is known exactly where the inserts have come from. This
process makes re-assembling the sequenced fragments to reflect their original position in the
genome easier and more accurate, and any one piece of human DNA sequence can automatically be
placed to an accuracy of 1 part in 30 000.
Each of the large clones is then 'shotgunned' - broken into pieces of perhaps 1500 base pairs,
either by enzymes or by physical shearing - and the fragments are sequenced separately.
Shotgunning the original large clone randomly several times ensures that some of the fragments will
overlap; computers then analyse the sequences of these small fragments, looking for end sequences
that overlap - indicating neighbouring fragments - and assembling the original sequence of the clone.
An alternative approach, 'whole genome shotgun sequencing', was first used in 1982 by the inventor
of shotgun sequencing, Fred Sanger, while working on phages (viruses of bacteria). As its name
suggests, in this technique the whole genome is broken into small fragments that can be sequenced
and reassembled. This method is very useful for organisms with smaller genomes, or when a related
genome is already known.
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Animal cloning
Animal clones are genetically identical. Natural clones occur in the form of
identical twins but it is also possible to produce artificial clones by nuclear transfer. The
nucleus is removed from a somatic (body) cell and placed in an egg whose own nucleus has
been removed. The egg is then implanted in a surrogate mother and develops to term.
Key principles
* Differentiated animal cells are unable to develop into complete animals
*The nuclei of most differentiated cells retain all the necessary genetic information.
* Transfer such a nucleus into an egg whose own nucleus has been removed.
* Transfer to the environment of the egg reprograms the nucleus (makes it forget its
history) and allows the full development of a viable animal that is genetically identical to
the donor of the somatic cell.
* Until 1997, cloning in mammals was only possible using nuclei obtained from very early
embryos. A breakthrough was made when cloning was achieved using nuclei from adult
cells.
* Recent research suggests that animals produced by cloning from adult cells may age
prematurely, but further investigation is necessary.
How does it work?
Nuclear transfer is carried out by fusing the donor somatic cell to an egg whose own
nucleus has been removed. Fusion is achieved in a culture dish by applying an electric
current. The change in electrical potential also mimics the normal events of fertilisation
and initiates development.
A key aspect in the success of nuclear transfer is synchronisation of the cell cycles
between the donor nucleus and the egg. Before fertilisation, the egg's nucleus is quite
inactive. The nucleus of the donor cell must also be made inactive otherwise it will not be
reprogrammed and development will fail. Inactivation is achieved by culturing the cell but
starving it of essential nutrients. The cell stops dividing and enters a quiescent state
compatible with nuclear transfer.
How is it used?
Animal cloning has the potential to overcome the limitations of the normal breeding cycle.
In the future, it may be used to produce elite herds by cloning the superior animals, or to
rapidly produce herds of transgenic or otherwise modified animals. Transgenic farm
animals make useful bioreactors, producing valuable proteins in their milk.
Another application is the use of genetically-modified pigs as a source of organs suitable
for transfer to humans (xenotransplantation).
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118
How is a specific gene isolated (CLONED)?
Its like going to the library and looking for a specific book.
It involves screening through a genomic library.
A genomic library is a large collection of plasmids containing
pieces of DNA from a specific species.
The set of cloned fragments is so comprehensive that virtually
the entire genome is represented in the library.
The fragments that make up the library are initially generated
by digesting genomic DNA (e.g. human) with a restriction
enzyme- say EcoRI
The EcoRI sites are randomly distributed in the genomefragments of varying lengths will be generated.
Some fragments will contain one gene, others two genes or cut
genes in half.
A
Gene1
Gene2
B
C
Gene3
D
E
F
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A
Gene1
Gene2
B
C
Gene3
D
E
F
Each fragment is cloned into the plasmid, each plasmid is put
(transformed) into E.coli
C
D
A
B
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The library is random!
Each fragment is cloned into the plasmid, each plasmid is put
(transformed) into E.coli
C
D
A
B
A
Gene1
Gene2
B
C
Gene3
D
E
F
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Fragments,bookmark, title
The library is not bookmarked or even titled and is in
fragments!
There is no organization to the library. It is simply a
populations of cloned fragments representing the entire genome.
The equivalent of this would be if you went to the University
Library to find all the books in a large heap, the books had no
title, and in addition instead of entire books you often found
parts of books.
How do you use such a library? How do you find the book you
are interested in.
Lets work our way through this problem with a simple example
Organism has EIGHT genes in its genome
A
B
C
D
E
F
G
H
EcoRI
122
Genomic library
If we wanted to study gene CCreate a restriction map of gene C
Determine it sequence
Study proteinC
What do we need to do
We need to initially clone the gene and make many copies of
gene C
Creating a genomic library provides a means of obtaining many
copies of gene C
To generate a genomic library:
Total genomic DNA is isolated from the species of interest
The DNA is cut with EcoRI
A
A
B
C
D
b
E
d
b
C
d
E
F
F
G
G
H
h
h
123
Genomic library
These genomic DNA fragments are mixed with a plasmid that
has been linearized at a single EcoRI site (say pUC18)
b
d
Ampr
Ori
F
G
h
h
d
Ori
Ampr
C
Ampr
b
E
Ampr
A
Ori
Ori
Both the plasmid and genomic DNA have been cut with EcoRI,
they have complementary sticky ends
|
G A A T T C
C T T A A G
|
124
Recombinant plasmid
This process where foreign DNA is joined to plasmid DNA is
called ligation
It results in recombinant plasmid (foreign DNA+plasmid)
Each plasmid has one foreign EcoRI fragment
Each foreign fragment is still present as only one copy! This is
not useful.
A
b
d
E
F
G
h
h
b
C
d
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How are genomic libraries used?
If we are interested in studying gene C, you need the plasmid
containing gene C
Having a genomic library means you have gene C, but where is
it? Which colony on the Petri dish contains gene C?
Genomic libraries are much more complex than the one
described for our hypothetical 8 gene organism
You need to identify one recombinant plasmid out of 100,000’s
present in a library.
Identifying and isolating a specific plasmid is called screening a
library.
This requires a probe
A probe is a sequence complementary to PART of the sequence
one wishes to pull out.
You radiolabel the probe and once labeled the probe is used to
identify the plasmid containing E. coli colony
How do we get the probe?
126
The genomic library and a specific probe enabled us to achieve
two goals
Out of the billions of base pairs in a large genome, we have
been able to identify a few 1000 base pairs that correspond
to a specific gene of interest.
In addition we were able to isolate this sequence on a
specifically engineered plasmid
That allows us to make large quantities of this rare sequence.
Genomic libraries are described in terms of average fragment
size and the number of plasmids that must be screened to
have the entire genome represented
To have a good probability (>99%) of identifying a given DNA
sequence (gene) present in the collection of plasmids (library).
The number of plasmids (colonies) that must be screened is a
function of the size of the genome of the species from which
the Library was constructed.
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