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Basic techniques
--- Nucleic acid hybridization
complementary strands will associate and form double
stranded molecules
--- DNA sequencing
Determining the array of nucleotides in a DNA molecule
--- Restriction Enzymes
These enzymes recognize and cleave DNA at specific
sequences
--- Blotting
Allows analysis of a single sequence in a mixture of nucleic
acids from a single individual
--- DNA cloning
This allows the isolation and generation of a large number
of copies of a given DNA sequence from a single individual
--- Transformation
Stably integrating a piece of DNA into the genome of an
organism
--- PCR
amplification (making many copies) of a known sequence
--- Genetic engineering
Altering the DNA sequence of a given piece of DNA
--- Genomics
Analyzing the entire genome OF INDIVIDUALS
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’ AAAAAATTTTAAAAA 3’
5’ AAAAAATTTTAAAAA 3’
Will associate with
Will NOT associate with
3’ TTTTTTAAAATTTTT 5’
3’ TTTTTTAAGATTTTT 5’
This occurs with complementary
DNA/DNA, DNA/RNA, RNA/RNA
2
Sequencing
Genomic DNA
Fragment DNA
(clone)
Sequence fragments
ACGCGATTCA
ACGCGATTCA
GCGATTCAGGTTA
GATTCAGGTTA
CAGGTTACCACGC
ACGCGTAGCGC
TAGCGCA
TAGCGCATTACAC
ACGCGATTCAGGTTACCACGCGTAGCGCATTACAC
Align fragments
Build consensus sequence
3
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
SHELBY
ACGCGGTTCAGGTTACCACGCGTAGCGCATTACAC
JAY
ACGCGATTCAGGTTACCACGCGTAAAACATTACAC
ADAM
ACGCGGTTCAGGTTACCCCGCGTAGCGCATTACAC
PAM
The sequence homology between Individuals is not perfect!!!
This allows us to assign a specific sequence to a specific
Individual
4
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
5
Restriction Enzymes
What are Restriction enzymes
What are restriction enzyme RECOGNITION 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
6
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 that recognizes
the sequence and cuts the sequence (but not in the
middle)
|
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
7
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
9
10
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
12
5’AAAAAAAAAAGGGGGGGGTTTTTTTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGGGGGTTTTTTTG
AATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAA
GTTTTTTTTCCCCCCCCAAAAAA5’
AATTCACGTACGTACGTACGTACGTACGTG
GTGCATGCATGCATGCATGCATGCACTTAA
5’AAAAAAAAAAGGGGTTTTTTTG
AATTCACGTACGTACGTACGTACGTACGTG
AATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTACCCCAAAAAAACTTAA
GTGCATGCATGCATGCATGCATGCACTTAA
GTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGTTTTTTTGAATTCACGTACGTACGTACGTACGTACGTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTACCCCAAAAAAACTTAAGTGCATGCATGCATGCATGCATGCACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’
13
Complementary sticky ends
AAAAAAGGGGGGGGTTTTTTTG
TTTTTTCCCCCCCCAAAAAAACTTAA
AATTCAAAAAAAAGGGGGGGGTTT3’
GTTTTTTTTCCCCCCCCAAA5’
AAAAAAGGGGGGGGTTTTTTTG AATTCAAAAAAAAGGGGGGGGTTT3’
TTTTTTCCCCCCCCAAAAAAACTTAA GTTTTTTTTCCCCCCCCAAA5’
GGCCCAAAAAAAAGGGGGGGGTTT3’
AAAAAAGGGGGGGGTTTTTTTG
GTTTTTTTTCCCCCCCCAAA5’
TTTTTTCCCCCCCCAAAAAAACTTAA
Enzyme compatibility
SmaI
AAACCCGGGAAA
TTTGGGCCCTTT
EcoRI
AAAGAATTCAAA
TTTCTTAAGTTT
XmaI
MfeI
AAACCCGGGAAA
TTTGGGCCCTTT
AAACAATTGAAA
TTTGTTAACTTT
AAAGAATTGAAA
TTTCTTAACTTT
Cant be cut by EcoRI or MfeI
KpnI
AAAGGTACCAAA
TTTCCATGGTTT
Asp718 AAAGGTACCAAA
TTTCCATGGTTT
SmaI
AAAAAACCCGGGAAAAAA
-----------------TTTTTTGGGCCCTTTTTT
XmaI
AAAAAACCCGGGAAAAAA
-----------------TTTTTTGGGCCCTTTTTT
EcoRI AAAAAAGAATTCAAAAAA
-----------------TTTTTTCTTAAGTTTTTT
MfeI
AAAAAACAATTGAAAAAA
-----------------TTTTTTGTTAACTTTTTT
KpnI
AAAAAAGGTACCAAAAAA
-----------------TTTTTTCCATGGTTTTTT
Asp718
AAAAAAGGTACCAAAAAA
-----------------TTTTTTCCATGGTTTTTT
xxxxxxx
17
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.
11Mu
vg
5Mu
cy
HindIII
EcoRI
205kb
300kb
Cy
PstI
Vg
Pr
100kb
SmaI
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.
18
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)
19
Sequence Divergence
The restriction map is a partial picture of the nucleotide sequence
of a piece of DNA.
By comparing restriction maps we can surmise differences in the
DNA sequence between species or between individuals
NNNNNNNNGAATTCNNNNNNNNNNNNNNNNAAGCTTNNNNNNNNNNNNNNCTGCAGNNNNNNNNNNNNNNN
NNNNNNNNCTTAAGNNNNNNNNNNNNNNNNTTCGAANNNNNNNNNNNNNNGACGTCNNNNNNNNNNNNNNN
GeneA
Mai California me rahta hu aur UCSC me padhata hu.
Human
Chimp
Gibbon
20
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.
21
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!!
22
Describing a DNA piece based on the pattern of restriction sites
Restriction map of a cloned piece of DNA
Linear or circular
E
H
E
B
K
S
B
How do I do this for an unknown piece of DNA?
B
S
K
B
Restriction map
Analogy
1
Goal: Identify the
parts for this blob
and describe the
spatial relationship
between the parts
2
a
c
You first Break the blob
apart into its separate
parts.
Look at the shapes and
number of the parts
b
3
a
c
b
Fit the parts together
To 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
26
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
7
Cut with EcoRI
5
4
1
8
2
2
Cut with HindIII
1
7
4
3
Cut with EcoRI and HindIII
1
3
5
2
27
EcoRI/HindIII
HindIII
HindIII
HindIII
2
Marker
EcoRI
5
EcoRI
3
EcoRI
EcoRI
1
Mapping unknown fragment
EcoRI 12kb 6kb and 2kb
HindIII
Marker
uncut
EcoRI
You are given a 20 kb linear fragment of DNA
After trying many enzymes you find that EcoRI and
HindIII cut the fragment
HindIII 14kb and 6kb
Solve the map
H
6
20
14
14
6
12
14
Or its mirror image
6
14
4
2
6
They are the same
1
What about EcoRI?
12
6
12
2
2
6
2
6
12
12
6
2
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?
A double digest with both enzymes will provide the answer
Marker
EcoRI
HindIII
EcoRI+HindIII
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
So HindIII site must be in the 12 kb
EcoRI fragment
14
12
6
4
2
1
2
6
12
8
4
8
6
6
H
6
4
2
2
H
8
8
H
4
2
4
HindIII alone gives 14 & 6
Which of the three maps gives 14 and 6 for HindIII alone?
29
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
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
31
EcoRI+ PstI
PstI
EcoRI+ HindIII
EcoRI
HindIII
Marker
Another linear map
14
12
8
6
4
2
1
32
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
33
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
34
Mapping
EcoRI
12
6
2
5
6
P
2
12
E
P
2
5 & 15
12
E
1 & 19
5
6
E
2 E
1
2
E
E
2
6
P
12
E
12
E
5
6
5
2
PstI/EcoRI
12
5
2
1
Pst I
1
6
1
PstI
13
7
6
3 & 17
12
E
P1
7 & 13
12
E
35
4
H
8
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
36
Xxxxxx
37
THE GENE PROBE!!!
How do we isolate a GENE?
How did we get a pure copy of the gene?
38
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
39
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
40
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
|
AATTCTTTTTTTTTTTAAAAAAGAATT
GAAAAAAAAAAATTTTTTCTTAA
AATTCAAAAAGGGGGTTTTTTTG
TTAAGTTTTTCCCCCAAAAAAACTTAA
5’AAAAAAAAAAGGGGTTTTTTTG
AATTCAAAAAAAAAAAAAAGGGGGGGGTTTTTTTG
AATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTACCCCAAAAAAACTTAA
GTTTTTTTTTTTTTTCCCCCCCCAAAAAAACTTAA
GTTTTTTTTCCCCCCCCAAAAAA5’
41
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
42
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
43
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.
44
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
45
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
46
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
47
Cloning DNA
When a piece of DNA is cut with EcoRI,
you get
|
GAATTC
CTTAAG
|
5’AAAAAAAAAAGGGGTTTTTTTG
AATTCAAAAAAAAAAAAAAGGGGGGGGTTTTTTTG
AATTCAAAAAAAAGGGGGGGGTTTTTT3’
3’TTTTTTTTTACCCCAAAAAAACTTAA
GTTTTTTTTTTTTTTCCCCCCCCAAAAAAACTTAA
GTTTTTTTTCCCCCCCCAAAAAA5’
5’AAAAAAAAAAGGGGTTTTTTTG
3’TTTTTTTTTACCCCAAAAAAACTTAA
AATTCAAAAAAAAAAAAAAGGGGGGGGTTTTTTTG
GTTTTTTTTTTTTTTCCCCCCCCAAAAAAACTTAA
When two pieces of DNA cut with EcoRI are ligated back
together you get back an EcoRI site
-----------------G AATTC---------------------------------CTTAA G-----------------
48
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
49
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
50
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
51
not useful.
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
52
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
53
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.
54
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
55
Applications of gene cloning
Sequencing cloned DNA
Mutagenizing cloned DNA at a specific site
Expression of foreign genes
Studying regulation of gene expression
Generating probes for FISH, southern blotting etc.
56
Cloning genes
Cloning and Expression of genes in cells
Coding region of protein
Enhancer, Promoter, Ribosome binding site
No cloning of RNA into
double stranded plasmid
DNA
No cloning of single
stranded DNA into double
stranded plasmid DNA
H
E B
Change promoter of gene
E
E
Protein
coding
Prm
58
E
H
E
E B
First clone Coding region
E
H
E
B
E
H
E
B
B
K
S
B
Promoter cloning
E
H
E
B
K
S
B
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
61
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
62
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.
The DNA present in the band corresponding to the insert can
be isolated from the gel
PURE GENE !!!!!
63
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
64
Definition of Key Terms
Traditional breeding
Conventional cross breeding of two species of plants to
transfer a gene from one species to the other (sexually
compatible)
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/animal with a gene
from a sexually incompatible plant/animal or other organism
Are cisgenics acceptable?
Are transgenics acceptable?
65
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 Genomic DNA and plasmid with restriction enzyme
Ligate with cut plasmid DNA
Grow individual plasmids in E. coli
Genomic Libraries
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
68
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
69
cDNA clone
RNA Cannot be cloned
So to clone RNA, you first convert RNA into DNA
Reverse transcriptase copies RNA into DNA
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 if you isolate RNA from blood cells, most of the
RNA is globin RNA.
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)
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cDNA synthesis
Protein coat
RNA genome
Reverse
transcriptase
mRNA
mRNA
DNA
RT
DNA
cDNA
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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
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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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Repeat- 95C55C70C
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5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
PRIMER1 5’AAAGATC3’
3’AGCTAGAT5’ PRIMER2
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’AGCTAGAT5’
5’AAAGATC3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
5’AAAGATCGGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCC AGCTAGAT5’
5’AAAGATC GGGGGGGGGGGGGGGTCGATCTA3’
3’TTTCTAGCCCCCCCCCCCCCCCAGCTAGAT5’
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PCR
How do you detect PCR?
Agarose Gels
Size of PCR product will depend upon location of PCR primers
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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?
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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).
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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?
87
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
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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
Indi 1
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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Individual3
Individual2
Individual1
Marker
Gene C
GeneC
From Individual1
Used as probe
92
E
E
1
Restriction mapping Individuals
E
4
E
2
Indi 1
Gene
A
E
1
E
1
B
E
E
4
Indi 2
Gene
Indi 1
Indi2
Indi1
Marker
Marker
B
Marker
Marker
A
Indi2
Mapping deletion with probe A. Mapping deletion with probe B.
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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
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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.
Individual 1
Globin probe
Kidney
Bone marrow
Brain
erythrocyte
Lymphocyte
Kidney
Bone marrow
Brain
erythrocyte
Lymphocyte
The RNA can be isolated from specific tissues
Individual 2
Globin probe
Presence of RNA is a reflection of gene activity
96
Gene3
Gene4
AAAAAAAAAA
TTTTTTTTTTT
GAGAGAGAGA
CTCTCTCTCT
GGGGGGGGG
CCCCCCCCCC
TATATATA
ATATATAT
TATATA
ATATATATAT
TTTTTTTTTT
CTCTCTCTCT
CCCCCCCCC
ATATATATAT
TATATA
ATATATATAT
CCCCCCCCC
GGGGGG
CCCCCCCCC
CTCTCTCTCT
GAGAGA
CTCTCTCTCT
TTTTTTTTTT
AAAAAA
TTTTTTTTTT
GGGGGG
GAGAGA
Gene2
AAAAAA
Gene1
CCCCCCCCC
GGGGGG
CCCCCCCCC
CCCCCCCCC
GAGAGA
AAAAAA
TTTTTTTTTT
TTTTTTTTTT
ATATATATAT
ATATATATAT
TATATA
ATATATATAT
TTTTTTTTTT
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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 Indi1 and
Indi2 cell,
Label each total RNA differently
Indi1=red
Indi2=green
Add labeled RNA from Indi1 and
Indi2 to array and let hybridize
Measure label and determine
change
Indi1 Indi2
1
2
3
4
5
Ratio of indi1/indi2
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1, 2, 3,4 … 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.
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