ABSTRACT
Polymerase chain reaction (PCR ; Figure 15.5 ) i s a powerfu l n e w techniqu e b y
w h i c h a targete d D N A sequenc e ca n b e exponentiall y amplifie d in vitro b y
repeated cycle s o f enzymati c D N A synthesis . Knowledg e o f th e sequenc e flank -
ing th e targete d regio n i s require d to construc t tw o D N A oligonucleotid e pr im -
ers complementar y t o th e D N A o n th e flank s o f th e targe t regions . Thes e
primers, 20-3 0 base s long , ca n hybridiz e t o opposit e strand s o f th e targe t se -
quence, ar e oriente d wi t h thei r 3 ' end s pointin g towar d eac h other , an d defin e
the end s o f th e D N A tha t wi l l b e replicated . I n th e firs t step , th e D N A t o b e
copied i s hea t denature d to separat e th e tw o complementar y D N A strands . Th i s
is followe d b y annealin g (hybridizing) th e primer s t o th e denature d flankin g
sequences. Th e primers are the n extende d alon g th e single-strande d D N A tem -
plates wi t h thermostabl e D N A polymeras e i n th e presenc e o f th e fou r deoxy -
nucleoside triphosphate s tha t yiel d double-strande d D N A . A t thi s poin t ther e
are tw o copie s o f th e targe t sequence . Th e three-ste p cycl e i s repeated , produc -
ing copie s o f th e origina l D N A an d copie s o f th e primer-define d targe t D N A .
Copies o f copie s contai n onl y th e targe t sequence , wherea s copie s o f th e origi -
