pcr
TRANSCRIPT
Amplifying DNA in Vitro: The
Polymerase Chain Reaction (PCR)
The polymerase chain reaction, PCR, can
produce many copies of a specific target
segment of DNA
A three-step cycle—heating, cooling, and
replication—brings about a chain reaction that
produces an exponentially growing population
of identical DNA molecules
Polymerase chain reaction
a.k.a. DNA amplification
in vitro method to specifically amplify nucleic acid
sequences
A most important and versatile technique
Very sensitive
Quick
Easy
robust
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From one… …to billions
of copies
Amplification by replicating
a specific sequence of DNA
many times
Fig. 20-8 5
Genomic DNA
TECHNIQUE
Cycle 1
yields
2
molecules
Denaturation
Annealing
Extension
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Target
sequence
Primers
New
nucleo-
tides
3
3
3
3
5
5
5 1
2
3
Fig. 20-8a
5
Genomic DNA
TECHNIQUE
Target
sequence
3
3 5
Fig. 20-8b
Cycle 1
yields
2
molecules
Denaturation
Annealing
Extension
Primers
New
nucleo-
tides
3 5
3
2
5 3 1
Fig. 20-8c
Cycle 2
yields
4
molecules
Fig. 20-8d
Cycle 3 yields 8
molecules; 2 molecules
(in white boxes)
match target sequence
DNA Amplification = DNA replication
Same requirements !
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Double helix must unwind
Primers
Deoxynucleotides (dNTPs)
Polymerase
allserv.rug.ac.be/~avierstr/ principles/pcr.html
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http://www.agen.ufl.edu/~chyn/age2062/lect/lect_09/lect_09.htm
Important to understand :
PCR is specific because of the primers
we design the primers to anneal to specific sequences
That means, we must know what we want to amplify
The sequence between the two primers is amplified other parts of the DNA will not be amplified
Amplification is exponential the newly synthesised DNA served as templates for further
rounds of amplification
The power of PCR
e.g. detection of viral /bacterial pathogens
Current technology
culture the pathogen
Run biochemical/ immunological test
Time consuming
Some viruses & bacteria cannot yet be cultured
- Delay diagnosis, wrong diagnosis, or not specific
enough
- e.g. SARS, bird flu
With PCR
Go straight for the DNA
No need to culture, biochemical test etc.
e.g Bacillus anthracis
First, we must be able to identify a gene/DNA sequence that is
unique to the anthrax bacteria
This gene or sequence must be present only in the B.anthracis
DNA, and not the DNA of any other organism, virus etc.
Then, we need to know a bit of the DNA sequence of this
anthrax gene (the ‘target’)
So that we can design specific primers that only anneals
to the target anthrax DNA
and not any other DNA
Then we do a PCR using these anthrax-specific primers
and DNA extracted from patient’s blood
Only a very small amount of blood is required < 0.1 ml
http://nobelprize.org/chemistry/laureates/1993/illpres/pcr.gif
+ve sample
Primers
anneal to
target DNA
anthrax
gene is
amplified
-ve sample
No amplification
Because
primers cannot
anneal with any
other DNA
Check for amplification products using a gel
PCR offers many advantages :
no need to isolate/culture the pathogens – extract DNA from soil and other samples
Very sensitive – amplify from only one copy of target (well, in theory), so requires very little sample
Highly specific – can even identify different species or strains accurately
Fast – a few hours
Allows for diagnosis before disease develops
This is only one of the many applications of PCR, we will discuss may more later
How to do
a PCR ?
What I need ?
PCR reaction requires
Target DNA – a tiny bit will do
Specific primers - most crucial
Must know sequence
Must be correctly designed DNA polymerase - heat stable
Thermocycler - machine to heat and cool
dNTPs – building blocks for new DNA synthesis
Buffer – to provide correct conditions for the enzyme to work
Typical reaction mix
Buffer + MgCl2
dNTPs – dATP,dCTP,dTTP,dGTP
Primers - forward and reverse
Polymerase
DNA sample
Thermocycling
Denaturation 94C
Annealing 55 C
Extension 72C
Buffer & MgCl2
Always use buffer that comes with enzyme
(unless you’re one of those who knows better!)
MgCl2 - affects specificity and yield
usually about 2 mM
High [MgCl] - more product but less specific
Low [MgCl] – less product but more specific
Optimise !
dNTPs
premade & premix – just buy them
Use 50 to 500 mM each
50 mM enough to make 6 ug of products
Primers
Most crucial
Primers are designed by you and synthesized on a machine
Occasionally primers fail for apparently no reason, so don’t feel bad
Ensure quality of primers – get a good supplier
Guidelines
Check orientation of primers
20 to 30 base pair long
Go for 40 to 50% GC content
Avoid internal structure
Avoid complimentarity between primers, esp at 3’ end
Avoid extensive GC’s at 3’ end
Orientation of primers
CTTATTAGTTTACTAT
5’CTTATTAGTTTACTATAAAGGAGTCGAAAGAGAAGTACCAAAGAT 3’
3’GAATAATCAAATGATATTTCCTCAGCTTTCTCTTCATGGTTTCTA 5’ .
CTCTTCATGGTTTCTA
. Forward primer = 5’CTTATTAGTTTACTAT 3’
Reverse primer = 5’ATCTTTGGTACTTCTC 3’
5’CTTATTAGTTTACTATAAAGGAGTCGAAAGAGAAGTACCAAAGAT 3’
3’CTCTTCATGGTTTCTA 5’
5’CTTATTAGTTTACTAT 3’
3’GAATAATCAAATGATATTTCCTCAGCTTTCTCTTCATGGTTTCTA 5’
Correct primers amplification
One wrong primer no amplification
Two wrong primers no amplification
Things to avoid
Internal structures
cattgccgacggcttaatcgta
a g=c
c=g c=g cattg=cttaatcgta Complementary 3’ ends
5’ cgtacgtactggttacctacgc 3’ | | | | | | |
3’ ggatgcgaattagactgacgc 5’
a ‘loop out’
‘primer dimer’
Polymerase
Taq polymerase from Thermus aquaticus
thermophilic - works at 72C
Others – Vent, Deepvent, TaKaRa, etc.
Polymerase makes error !
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Taq – no proof-reading activity – doesn’t correct error
Also add an extra base – A – at the ends
A
A
Error rate can be as high as one mistake in 1000
New generation of ‘proof reading enzymes’ –
e.g. Pfu, Pfx, Pwo
has 3’exonuclease and proofreading activity
Extra A at the end – can be used in T/A cloning systems
Applications of PCR
Medical
Forensics (CSI)
Detection of infectious agents
Viral infections
Bioweapons
Difficult-to-culture organism or slow growing
Forensic Evidence and Genetic
Profiles
An individual’s unique DNA sequence, or genetic
profile, can be obtained by analysis of tissue or body
fluids
Genetic profiles can be used to provide evidence in
criminal and paternity cases and to identify human
remains
Genetic profiles can be analyzed using RFLP analysis
by Southern blotting
Even more sensitive is the use of genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences
PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
The probability that two people who are not identical twins have the same STR markers is exceptionally small
Fig. 20-24 This photo shows Earl Washington just before his release in 2001, after 17 years in prison.
These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder.
(a)
Semen on victim
Earl Washington
Source of sample
Kenneth Tinsley
STR marker 1
STR marker 2
STR marker 3
(b)
17, 19
16, 18
17, 19
13, 16
12, 12
14, 15
11, 12
13, 16
12, 12
Time to watch a movie….
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Reverse Transcriptase PCR for the detection of RNA viruses
(almost all of the most nasty viruses have RNA genomes –Ebola,
dengue, nipah, SARS – you name it)
- before PCR – reverse transcribed viral RNA to cDNA first
DNA fingerprinting
DNA fingerprinting using VNTRs
http://homepages.strath.ac.uk/~dfs97113/BB310/Lect1603.html
VNTRs - variable number of tandem repeats – a.k.a minisatellites
– natural polymorphisms in the human genome
Different numbers of a short, repeated sequence
Each repeat 15 – 100 bp long;
Repeated in tandem arrays up to 40 kb long
SSTR – simple sequence tandem repeats – a.k.a microsatellites
Repeats of 2 to 4 nucleotides
e.g. CAGCAGCAGCAGCAGCAGCAG daddy
CAGCAGCAGCAG mommy
daddy
mommy
VNTRs are hypervariable – can be very different between individuals
Many different types of VNTRs
-can be found at many loci in the genome
-two individual may have similar VNTRs at one loci
-But the chances of two individuals having the same pattern of VNTRs
at several loci is very small
The DNA sequences next to VNTRs are usually highly conserved
(very similar in every individual)
So we can design PCR primer to target these flanking sequences
Using these primers, we can amplify the VNTR regions
The VNTR amplification products will have different sizes, and can
be separated on an agarose or polyacrylamide gel
By using PCR to amplify all three regions, a unique
fingerprint can be generated for each individual
Fingerprinting by PCR of VNTR/STR
www-ermm.cbcu.cam.ac.uk/ 99000587h.htm
Family trees by DNA finger printing
http://www.people.virginia.edu/~rjh9u/vntr1.html
DNA fingerprinting
DNA fingerprinting
Real time PCR – the next evolution
Monitoring of amplification reaction in real time
Quantitative
Rapid - results-as-you –wait
Close system – minimal cross contamination
Much more expensive…………..
Detection of GMO’s
Most genetically modified plants contain the 35S promoter of
cauliflower mosaic virus (CaMV)
and the 3’ untranslated region (terminator) ofthe nopaline
synthase (NOS) gene of Agrobacterium tumefaciens.
Can be detected using specific primers
Detection using SYBRO Green or fluorescence molecular beacons
SYBRO Green fluoresces when bind to double stranded DNA only
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Detection of GM soya and maize by RT PCR