PCR PPT
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PCR
Polymerase Chain Reaction
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Invented by Kary Mullis
Mullis and Faloona, 1987. Specific synthesis of DNA in vitro via a
polymerase-catalyzed chain reaction.
Nobel Prize 1993
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Kary Mullis himself….
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“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
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PCR
Specifically targets and amplifies a SINGLE sequence from within a complex
mixture of DNA.
How is this different from cloning?
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Takes advantage of basic requirements of replication
A DNA template NucleotidesPrimerspolymerase
PCR is DNA replication in a test tube
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PrimersMust have some information about sequence flanking your target
Primers provide specificity
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5’
5’
3’
3’
Complementary to opposite strands with 3’ ends pointing towards each other
Should have similar melting temperatures
Be in vast excess
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Melting temperature
TmoC = 2(A/T) + 4(G/C)
TmoC Temperature at which
half possible H bonds are formed
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The basic process
dsDNA
Denature (95 degrees)
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5’
5’
3’
3’
http://www.dnalc.org/shockwave/pcranwhole.html
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Thermocycling
94 degrees55 degrees70 degree
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Heat-stable polymerase is vital to the ease of the process…
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Thermus aquaticus:
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The Thermus aquaticus DNA polymerase
Taq
Not permanently destroyed at 94ºCOptimal temperature is 72ºC
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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
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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
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Templates for PCR
Dried bloodSemen stains
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Templates for PCRDried bloodSemen stainsVaginal swabsSingle hairFingernail scrapingsInsects in AmberEgyptian mummiesBuccal SwabToothbrushes
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PCR variations
Add 5’ extensions for cloning5’ 3’
5’3’
TC
A
3’
3’
5’
AA
T
G
TC
5’
TA
G
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Cloning PCR Fragments
Taq leaves 3’ A overhang.
AA
TT
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rtPCR
Reverse trancriptase PCR
Use mRNA as a template
Isolated cDNA clones
Can be quantitative
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Inverse PCR
known unknown
Knownunknown unknown
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known
unknown
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Nested primers
PCR primers are not always an exact match!
Degeneracy
Lower annealing temperatures increase chances of amplifying something!
Could be wrong thing!
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Nested primers
1
12
2
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Quantitative PCR
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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
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SYBRgreen
Also binds primer dimersCan overestimate product
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Molecular Beacons
Uses FRETFuorescence Resonance Energy TransferUses two sequence specificoligonucleotides labeled with fluorescent dyes
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Taq Man
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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
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Other application of PCR
Preimplantation diagnosis of genetic diseasesForensicsPaternity testing
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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)
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STR analysis of family of last Tsar of Russia
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VNTR’s
Can use PCR to visualize VNTRs
Eg. pMCT118 in chromosome 1
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VNTR analysis
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Problems with PCR
ContaminationTheoretically one molecule can amplifyTakes one mismatch early on to amplify the wrong fragment