How to identify a clone from a DNA library

1
Lecture 7 How to identify a clone from a DNA
library Colony hybridisation Immunoscreening Compl
ementation of mutant phenotype The Polymerase
Chain Reaction Principle Primer design Cycling
profile Problems with mutation rate Application R
T-PCR Using degenerated primers to clone
homologue Cloning of PCR products
2
Learning outcomes After this lecture you
should be able to explain how a DNA library can
be used to isolate your favourite gene or piece
of DNA describe in detail how PCR can be used to
amplify DNA and which factors are important for a
successful PCR reaction
3
How to find your favourite gene in a DNA
library when you know part of the
sequence.... when you know a sequence from a
homologous gene in a different organism.... Use
DNA to identify clone in DNA library colony
hybridisation when you know the protein raise
antibodies Use antibodies to identify clone in
(cDNA) expression library immunoscreening
when you have a mutant Transform expression
library into mutant and look for rescue of
phenotype rescue by complementation
4
DNA can be radioactively labelled and used as a
probe to detect clone of interest Example
random prime labelling
Denature Add random hexamer primers
Klenow Polymerase dNTP
C
T
A
G
A
T
C
C
G
C
A
G
G
T
A









C
T













DNA label can be radioactive or non-radioactive
(biotin)
5
Identification of a DNA clone in a library by
hybridisation principle complementary DNA
strands hybridise to each other
At low temperature Hybridisation
raise temperature (wash)
Only specific complementary probe remains bound
6
Principle of colony hybridisation
replica onto nitrocellulose
NaOH Protease
Lyse cells denature DNA
pick colony and isolate plasmid
Incubate with radioactive probe Wash
Expose to X-ray film
7
Immunoscreening using expression library, vector
contains promotor
replica onto nitrocellulose
Lyse cells with chloroform
pick colony and isolate plasmid
Add antibody
Add radioactive protein A
Expose to X-ray film
8
Use an expression library to identify genes that
complement a mutant phenotype
trp-
needs tryptophan to survive
transform with expression library
grows on media without tryptophan
One plasmid contains trp gene
pick colony and isolate plasmid
9
Polymerase Chain Reaction (PCR) Alternative in
vitro way to amplify DNA Can be used to amplify
DNA in between two short oligonucleotide
sequences (primers) Fast and easy (hours in
stead of days/weeks) Popularity due to increased
number of sequenced genomes Can be combined with
classical cloning approach Limitations need
to know sequence to design primers maximum size
of product 40 kb relatively high rate of
nucleotide misincorporation
10
Principle of PCR
Denature at 94 C
5'
3'
3'
5'
Anneal primers at 50 - 60 C
repeat 30 times
5'
3'
3'
5'
Synthesise DNA at 72 C
5'
3'
3'
5'
11
The power of PCR Amplification of specific
sequence using 30 amplification
cycles 2301x109
12
Components of a PCR reaction Template
DNA Buffer with magnesium dATP, dTTP, dCTP and
dGTP (dNTP's) pair of primers (in excess of
template DNA) Polymerase Normal DNA
polymerase will denature at high temperatures
used for PCR. DNA polymerase found and isolated
from thermophile bacterium Thermus aquaticus,
lives in hot springs Half life at 95 C 1.6
hours
13
PCR primer design primer length Key to
successful PCR!!! Primers are chemically
synthesised, just order from company Length
short primers can anneal randomly...... length
of primer random primes in human
genome 8 46000 12 178 20
0.003 But long primers take longer to anneal,
reduces efficiency of PCR reaction In practice
primers are between 17 and 22 bp
14
PCR primer design product length Product length
is determined by distance between
primers Keep it short (if
possible)!!!! Between 20 bp and 1 kb is no
problem Between 1 kb and 10 kb can be a
problem Above 10 kb often is a problem.......
Product length
15
PCR primer design polarity and
orientation Points to consider primer needs to
be complementary to its template strand primer
is extended from 5' to 3' 3' ends should point
towards each other 5'cgtcgaactcgatcgatcgata-----
agctgatcgaactctatgcgc3'
3'cgactagcttgagatac5'
5'tcgaactcgatcgatcg3' 3'gcagcttgagctagctagctat----
-tcgactagcttgagatacgcg5' Convention write
primers from 5' to 3' (or clearly mark 5' and 3'
ends) Example catagagttcgatcagc tcgaactcgatcga
tcg
16
PCR primer design sequence Avoid sequence
complementarity within and between
primers 3' terminal nucleotide should
preferably a G or C (stronger hydrogen bond)
5'
3'
5'
3'
5'
3'
17
PCR primer design melting temperature Melting
temperature is dependent on primer length and GC
content Estimate melting temperature of
primer Tm4xGC 2xAT C Example
cgattatcgctatgcgc 9x4 8x2 52 C Optimal
annealing temperature of primer is a few (2-5)
degrees below melting temperature Melting
temperature of primers needs to match
18
Exercise primer design Which primer combination
would you use to amplify the sequence between the
following DNA fragments atcgcgatcgatctaggcggcta
cgtcgtttag.....gtagctcgtactcggctagtatctcgactgcgct
A tcgcgatcgatctaggcggc and cggctagtatctcgactgc
g B tcgcgatcgatctaggcggc and cgcagtcgagatacta
gccg C gccgcctagatcgatcgcgt and
cgcagtcgagatactagccg D gccgcctagatcgatcgcgt
and cggctagtatctcgactgcg Note Template DNA and
primers are written down in the 5' to 3'
orientation!!!
19
Exercise primer design Which primer combination
would you use to amplify the sequence between the
following DNA fragments atcgcgatcgatctaggcggcta
cgtcgtttag.....gtagctcgtactcggctagtatctcgactgcgct
A tcgcgatcgatctaggcggc and cggctagtatctcgactgc
g B tcgcgatcgatctaggcggc and cgcagtcgagatacta
gccg C gccgcctagatcgatcgcgt and
cgcagtcgagatactagccg D gccgcctagatcgatcgcgt
and cggctagtatctcgactgcg Note Template DNA and
primers are written down in the 5' to 3'
orientation!!!
20
Working out the optimal temperature cycling
profile
Denaturation
100
90
80
repeat 30 times.........
Extension
70
60
Temperature ( C)
50
Annealing
40
30
20
10
1
2
3
4
5
6
Time (minutes)
21
Working out the optimal temperature cycling
profile Denaturation Balance between optimal
denaturation (high temperature, long time) and
preservation of Taq (lower temperature, short
time) Typically 1' at 94 C Annealing Depende
nt on primers. Balance between efficiency (lower
temperature) and specificity (higher
temperature) Typically 1' at 2C 5C below
melting temperature Extension Balance between
efficiency and preservation of TAQ Typically 72
C, 1' per kb product
22
Problems with error rate of Taq polymerase All
polymerases mis-incorporate nucleotides Most
polymerases have proofreading mis-incorporated
nucleotide is removed by exonuclease activity and
correct nucleotide is inserted But Taq
polymerase lacks proofreading activity Alternativ
e polymerase from Pyrococcus furiosus (Pfu)
contains proofreading activity. But slow
polymerisation, less processive Polymerase Erro
r rate mutant molecules (mutation
frequency/bp/duplication) (30 cycles, 3
kb) Taq 8.0 x 10-6 51 Pfu 1.3 x
10-6 11
23
PCR application Reverse Transcription PCR
(RT-PCR) Use of PCR to amplify specific cDNA
from mRNA molecules
AAAAAAAA
AAAAAAAA
add oligo(dT) primer reverse transcriptase
TTTTTTTT
mRNA
RNA/DNA hybrid
RNase H
RNA Degradation
TTTTTTTT
Add primers PCR components
1st PCR cycle
Standard PCR
24
PCR application PCR with degenerated
primers When the protein sequence is known
(purified and sequenced protein or strongly
conserved between different organisms), but the
DNA sequence is not.......... M T F K L
P...........L P G K G D ATG ACT TTT
AAA TTA CCT TTA CCT GGT AAA GGT GAT
C C G C G C C G C C G C
C A T A T A A
A G C G C G G
G
PCR using mix of degenerated primers
Clone and sequence
25
Cloning of PCR products 1. introduce restriction
site in primer
5'
cgcgcgcttaag
3'
cgatatgatgcgactcg ...t
tacggcgatatacgactacgtcgtgctatactacgctgagctgattacgt
actgcgtgactgact...
3'
5'
PCR
cgcgcgcttaagcgatatgatgcgactcgactaatg
catgacgcactgactga...
gcgcgcgaattcgctatactacgctgagctgattacgtactgcgtgactg
act...
Restriction digestion
gcgatatgatgcgactcgactaatg
catgacgcactgactga...
aattcgctatactacgctgagctgattacgtactgcgtgactgact...

cgcgcgcttaa gcgcgcg
26
Cloning of PCR products 2. TA cloning Principle
Taq polymerase often adds an A to the 3' end of
the PCR product atcgcgttacgtacg..........cgattag
cgctagca atagcgcaatgcatgc..........gctaatcgcgatcg

Ligate
t a
t a
t
t
a
a
27
Cloning of PCR products 3. Blunt
cloning Principle proof reading polymerases
(e.g.Pfu) often leave blunt DNA ends
Ligate
28
So far...... Lecture 4/5 Introduction to the
use of cloning tools Lecture 6 How these tools
are used to create a DNA library Lecture 7 How
DNA libraries are used to find your favourite
gene/protein How PCR can be used to amplify DNA
Lecture 8 Sequencing and mutagenesis of cloned
genes