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A DNA template
Nucleotides
Primers
polymerase
3’ 5’
Be in vast excess
Melting temperature
3’ 5’
http://www.dnalc.org/shockwave/pcranwhole.html
Thermocycling
94 degrees
55 degrees
70 degree
Heat-stable polymerase is vital
to the ease of the process…
Thermus aquaticus:
The Thermus
aquaticus DNA
polymerase
Taq
Not permanently
destroyed at 94ºC
Optimal
temperature is 72ºC
Problems with Taq
Does not have proof readng ability
Error rate 1 in 2 X 104 bases
Seems rare but can be recovered in
cloning a single molecule
Newer polymerases have high fidelity
Termplates for PCR
Small amount of template
In theory a single molecule
Do not need to isolate sequence of
interest
DNA template need not be highly
purified
DNA is stable in absence of nucleases
Templates for PCR
Dried blood
Semen stains
Templates for PCR
Dried blood
Semen stains
Vaginal swabs
Single hair
Fingernail scrapings
Insects in Amber
Egyptian mummies
Buccal Swab
Toothbrushes
PCR variations
Add 5’ extensions for cloning
5’ 3’
5’ 3’ 5’
G
A
A
T
T 3’
C
3’ C
T
T
A
A
G 5’
Cloning PCR Fragments
Taq leaves 3’ A overhang.
A
A
T
T
rtPCR
Reverse trancriptase PCR
Can be quantitative
Inverse PCR
known unknown
unknown
Nested primers
Degeneracy
1 2
2 1
Quantitative PCR
Real Time PCR
Detection and quantitation of fluorescent reporter the
signal of which increases in direct proportion to the
amount of PCR product in a reaction
Also binds
primer dimers
Can
overestimate
product
Molecular Beacons
Uses FRET
Fuorescence Resonance Energy Transfer
Uses two sequence specific
oligonucleotides labeled with fluorescent
dyes
Taq Man
Other application of PCR
Detection of mutations
screen for inherited disorders
Detection of HIV
Not standard test given
Detect tuberculosis without culturing
Prenatal sex determination
DZY1 = Y specific sequence present in
5000 copies
Other application of PCR
Preimplantation diagnosis of genetic
diseases
Forensics
Paternity testing
Forensics
STR
Short Tandem Repeats
2 to 7 base pairs repeated 7-40 times
Replaced VNTRs in forensic analysis
13 Highly polymorphic loci have been
selected by FBI
Population match probabilities 0.1 - 0.28
Probability One in 5.7 X 10-15