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 NucleotidesPrimerspolymerase

PCR is DNA replication in a test tube

PrimersMust have some information about sequence flanking your target

Primers provide specificity

5’

5’

3’

3’

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’

5’

3’

3’

http://www.dnalc.org/shockwave/pcranwhole.html

Thermocycling

94 degrees55 degrees70 degree

Heat-stable polymerase is vital to the ease of the process…

Thermus aquaticus:

The Thermus aquaticus DNA polymerase

Taq

Not permanently destroyed at 94ºCOptimal temperature is 72ºC

Problems with Taq

Does not have proof readng ability Error rate 1 in 2 X 104 basesSeems rare but can be recovered in cloning a single moleculeNewer polymerases have high fidelity

Termplates for PCR

Small amount of templateIn theory a single moleculeDo not need to isolate sequence of interestDNA template need not be highly purifiedDNA is stable in absence of nucleases

Templates for PCR

Dried bloodSemen stains

Templates for PCRDried bloodSemen stainsVaginal swabsSingle hairFingernail scrapingsInsects in AmberEgyptian mummiesBuccal SwabToothbrushes

PCR variations

Add 5’ extensions for cloning5’ 3’

5’3’

TC

A

3’

3’

5’

AA

T

G

TC

5’

TA

G

Cloning PCR Fragments

Taq leaves 3’ A overhang.

AA

TT

rtPCR

Reverse trancriptase PCR

Use mRNA as a template

Isolated cDNA clones

Can be quantitative

Inverse PCR

known unknown

Knownunknown 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

12

2

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

SYBRgreen

Also binds primer dimersCan overestimate product

Molecular Beacons

Uses FRETFuorescence Resonance Energy TransferUses two sequence specificoligonucleotides labeled with fluorescent dyes

Taq Man

Other application of PCR

Detection of mutationsscreen for inherited disorders

Detection of HIVNot standard test given

Detect tuberculosis without culturingPrenatal sex determination

DZY1 = Y specific sequence present in 5000 copies

Other application of PCR

Preimplantation diagnosis of genetic diseasesForensicsPaternity testing

ForensicsSTRShort Tandem Repeats

2 to 7 base pairs repeated 7-40 timesReplaced VNTRs in forensic analysis

13 Highly polymorphic loci have been selected by FBI

Population match probabilities 0.1 - 0.28Probability 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

ContaminationTheoretically one molecule can amplifyTakes one mismatch early on to amplify the wrong fragment