polymerase chain reaction. invented by kary mullis mullis and faloona, 1987. specific synthesis of...

Polymerase Chain Reaction

Invented by Kary Mullis

1048708 Mullis and Faloona 1987 Specificsynthesis of DNA in vitro via apolymerase-catalyzed chain reaction

1048708 Nobel Prize 1993

ldquoI was working for Cetus making oligonucleotidesThey were heady times Biotechnology was in flowerand one spring night while the California buckeyeswere also in flower I came across the polymerasechain reaction I was driving with Jennifer Barnett toa cabin I had been building in northern CaliforniaShe and I had worked and lived together for twoyears She was an inspiration to me during that timeas only a woman with brains in the bloom of herwomanhood can be That morning she had no ideawhat had just happened I had an inkling It was thefirst day of the rest of my liferdquo- from Karry Mullisrsquos autobiography at the Nobel e-Museum

Specifically targets and amplifies aSINGLE sequence from within a complexmixture of DNAHow is this different from cloning

Takes advantage of basicrequirements of replication1048708 A DNA template1048708 Nucleotides1048708 Primers1048708 polymerasePCR is DNA replication in a test tube

Primers1048708 Must have some information aboutsequence flanking your target1048708 Primers provide specificity

1048708 Complementary to opposite strands with 3rsquoends pointing towards each other1048708 Should have similar melting temperatures1048708 Be in vast excess

Melting temperature

1048708 TmoC = 2(AT) + 4(GC)1048708 TmoC Temperature at whichhalf possible H bonds areformed

Steps of PCR

Annealing then primer elongation

Thermocycling

1048708 94 degrees

1048708 55 degrees

1048708 70 degree

Heat-stable polymerase is vitalto the ease of the processhellip

Problems with Taq

Automated long time agohellip

Problems with PCR

1048708 Contamination

1048708 Takes one mismatch early on to amplify the wrong fragment