Sunteți pe pagina 1din 42

PCR

Polymerase Chain Reaction


Invented by Kary Mullis
„ Mullis and Faloona, 1987. Specific
synthesis of DNA in vitro via a
polymerase-catalyzed chain reaction.

„ Nobel Prize 1993


Kary Mullis himself….
“I was working for Cetus, making oligonucleotides.
They were heady times. Biotechnology was in flower
and one spring night while the California buckeyes
were also in flower I came across the polymerase
chain reaction. I was driving with Jennifer Barnett to
a cabin I had been building in northern California.
She and I had worked and lived together for two
years. She was an inspiration to me during that time
as only a woman with brains, in the bloom of her
womanhood, can be. That morning she had no idea
what had just happened. I had an inkling. It was the
first day of the rest of my life.”
- from Karry Mullis’s autobiography at the Nobel e-Museum
PCR

Specifically targets and amplifies a


SINGLE sequence from within a complex
mixture of DNA.

How is this different from cloning?


Takes advantage of basic
requirements of replication

„ A DNA template
„ Nucleotides
„ Primers
„ polymerase

PCR is DNA replication in a test tube


Primers
„ Must have some information about
sequence flanking your target

„ Primers provide specificity


5’ 3’

3’ 5’

„ Complementary to opposite strands with 3’


ends pointing towards each other

„ Should have similar melting temperatures

„ Be in vast excess
Melting temperature

„ TmoC = 2(A/T) + 4(G/C)

„ TmoC Temperature at which


half possible H bonds are
formed
The basic process
dsDNA

Denature (95 degrees)


5’ 3’

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

„ Use mRNA as a template

„ Isolated cDNA clones

„ Can be quantitative
Inverse PCR

known unknown

unknown Known unknown


known

unknown
Nested primers

PCR primers are not always an exact match!

Degeneracy

Lower annealing temperatures increase chances of


amplifying something!

Could be wrong thing!


Nested primers

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

Does not measure the amount of end product but its


production in real time
SYBR
green

„ 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

„ Combined DNA Index System (CODIS)


STR analysis of family of last
Tsar of Russia
VNTR’s
„ Can use PCR to visualize VNTRs

„ Eg. pMCT118 in chromosome 1


VNTR analysis
Problems with PCR
„ Contamination
„ Theoretically one molecule can amplify
„ Takes one mismatch early on to amplify
the wrong fragment