Post on 21-Dec-2014
description
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