POLYMERASE CHAIN REACTION: METHODS, PRINCIPLES

AND
APPLICATION
Dr.Mohini Joshi1*, Dr.Deshpande |.D2.
The poymerase chan reacton (PCR) s a scentc technque n
moecuar boogy to ampfy a snge or a few copes of a pece
of DNA across severa orders of magntude, generatng
thousands to mons of copes of a partcuar DNA sequence.
PCR s now a common and often ndspensabe technque used
n medca and boogca research abs for a varety of
appcatons. There are three ma|or steps nvoved n the PCR
technque: denaturaton, anneang, and extenson. PCR s
usefu n the nvestgaton and dagnoss of a growng number
of dseases. Ouatatve PCR can be used to detect not ony
human genes but aso genes of bactera and vruses. PCR s
aso used n forenscs aboratores and s especay usefu
because ony a tny amount of orgna DNA s requred. PCR can
dentfy genes that have been mpcated n the deveopment of
cancer. Moecuar conng has beneted from the emergence of
PCR as a technque. The present paper s an attempt to revew
bascs of PCR.
Introducton
The poymerase chan reacton (PCR) s a scentc technque n
moecuar boogy to ampfy a snge or a few copes of a pece
of DNA across severa orders of magntude, generatng
thousands to mons of copes of a partcuar DNA sequence.
Poymerase Chan Reacton was deveoped n 1984 by the
Amercan bochemst, Kary Mus. Mus receved the Nobe
Prze and the |apan Prze for deveopng PCR n 1993.1 However
the basc prncpe of repcatng a pece of DNA usng two
prmers had aready been descrbed by Gobnd Khorana n
1971. Progress was mted by prmer synthess and poymerase
purcaton ssues.2 PCR s now a common and often
ndspensabe technque used n medca and boogca
research abs for a varety of appcatons.3 The poymerase
chan reacton s a powerfu technque that has rapdy become
one of the most wdey used technques n moecuar boogy
because t s quck, nexpensve and smpe. The technque
ampes specc DNA fragments from mnute quanttes of
source DNA matera, even when that source DNA s of
reatvey poor quaty.4 PCR; the quck, easy method for
generatng unmted copes of any fragment of DNA, s one of
those scentc deveopments that actuay deserve tmeworn
superatves ke "revoutonary" and "breakthrough. From the
day practcates of medca dagnoss to the theoretca
framework of systematcs, from courts of aw to ed studes of
anma behavor, PCR takes anayss of tny amounts of genetc
matera-even damaged genetc matera to a new eve of
precson and reabty. Furthermore, many mportant
contrbutons to the deveopment and appcaton of PCR
technoogy have been made; however the present paper s an
attempt to revew bascs of PCR.
Basc Concept of PCR
The basc PCR prncpe s smpe. As the name mpes, t s a
chan reacton: One DNA moecue s used to produce two
copes, then four, then eght and so forth. Ths contnuous
doubng s accompshed by specc protens known as
poymerases, enzymes that are abe to strng together
ndvdua DNA Budng bocks to form ong moecuar strands.
To do ther |ob poymerases requre a suppy of DNA budng
bocks, .e. the nuceotdes consstng of the four bases adenne
(A), thymne (T), cytosne (C) and guanne (G). They aso need a
sma fragment of DNA, known as the prmer, to whch they
attach the budng bocks as we as a onger DNA moecue to
serve as a tempate for constructng the new strand. If these
three ngredents are supped, the enzymes w construct exact
copes of the tempates. PCR s a method used to acqure many
copes of any partcuar strand of nucec acds. Its a means of
seectvey ampfyng a partcuar segment of DNA. The
segment may represent a sma part of a arge and compex
mxture of DNAs e.g. a specc exon of a human gene. It can be
thought of as a moecuar photocoper. PCR can ampfy a
usabe amount of DNA (vsbe by ge eectrophoress) n -2
hours. The tempate DNA need not be hghy pured a boed
bactera coony. The PCR product can be dgested wth
restrcton enzymes, sequenced or coned. PCR can ampfy a
snge DNA moecue, e.g. from a snge sperm. The poymerase
chan reacton rees on the abty of DNAcopyng enzymes to
reman stabe at hgh temperatures. PCR has transformed the
way that amost a studes requrng the manpuaton of DNA
fragments may be performed as a resut of ts smpcty and
usefuness.5 In Mus's orgna PCR process, the enzyme was
used n vtro. The doube-stranded DNA was separated nto two
snge strands of DNA by heatng t to 96C. At ths
temperature, however, the E.Co DNA poymerase was
destroyed, so that the enzyme had to be repenshed wth new
fresh enzyme rafter the heatng stage of each cyce. Mus's
orgna PCR process was very nemcent snce t requred a
great dea of tme, vast amounts of DNA-Poymerase, and
contnua attenton throughout the PCR process.
Steps n PCR
There are three ma|or steps nvoved n the PCR technque:
denaturaton, anneang, and extenson.
In step one; the DNA s denatured at hgh temperatures (from
90 - 97 degrees Cesus).
In step two, prmers annea to the DNA tempate strands to
prme extenson.
In step three, extenson occurs at the end of the anneaed
prmers to create a compementary copy strand of DNA.
Ths ehectvey doubes the DNA quantty through the thrd
steps n the PCR cyce. To ampfy a segment of DNA usng PCR,
the sampe s rst heated so the DNA denatures, or separates
nto two peces of snge-stranded DNA. Next, an enzyme caed
"Taq poymerase" syntheszes buds two new strands of DNA,
usng the orgna strands as tempates. Ths process resuts n
the dupcaton of the orgna DNA, wth each of the new
moecues contanng one od and one new strand of DNA. Then
each of these strands can be used to create two new copes,
and so on, and so on.7 The anneang phase happens at a ower
temperature, 50-60C. Ths aows the prmers to hybrdze to
ther respectve compementary tempate strands, a very usefu
too to forensk chemstry. The newy-formed DNA strand of
prmer attached to tempate s then used to create dentca
copes oh the orgna tempate strands desred. Taq
poymerase adds avaabe nuceotdes to the end of the
anneaed prmers. The extenson of the prmers by Taq
poymerase occurs at approx 72C for 2-5 mnutes. DNA
poymerase I cannot be used to eongate the prmers as one
woud expect because t s not stabe at the hgh temperatures
requred for PCR. The beauty of the PCR cyce and process s
that t s very fast compared to other technques and each
cyce doubes the number of copes of the desred DNA strand.
After 25-30 cyces, whoever s performng the PCR process on a
sampe of DNA w have penty of copes of the orgna DNA
sampe to conduct expermentaton. Assumng the maxmum
amount of tme for each step, 30 cyces woud ony take 6
hours to compete.
As the process of denaturaton, anneang, and poymerase
extenson s contnued the prmers repeatedy bnd to both the
orgna DNA tempate and compementary stes n the newy
syntheszed strands and are extended to produce new copes of
DNA. The end resut s an exponenta ncrease n the tota
number of DNA fragments that ncude the sequences between
the PCR prmers, whch are nay represented at a theoretca
abundance of 2n, where n, s the number of cyces.
Due to the ntroducton of a thermostabe DNA poymerase, the
Taq DNA poymerase once, at the begnnng of the PCR
reacton.9 The thermostabe propertes of the DNA poymerase
actvty were soated from Thermus aquatcus (Taq) that grow
n geysers of over 110C, and have contrbuted greaty to the
yed, speccty, automaton, and utty of the poymerase
chan reacton. The Taq enzyme can wthstand repeated heatng
to 94C and so each tme the mxture s cooed to aow the
ogonuceotde prmers to bnd the catayst for the extenson s
aready present.10 After the ast cyce, sampes are usuay
ncubated at 72C for 5 mnutes to n the protrudng ends of
newy syntheszed PCR products. To ensure success, care
shoud be taken both n preparng the reacton mxture and
settng up the cycng condtons. Increasng the cyce number
above -35 has tte postve ehect because the pateau occurs
when the reagents are depeted; accumuate. The speccty of
ampcaton depends on the extent to whch the prmers can
recognze and bnd to sequences other than the ntended target
DNA sequences.
Basc Technques n Moecuar Boogy
Dr. Stefan Surzyck.
PCR Analysis
The Poymerase Chan Reacton (PCR) s a powerfu method of
n vtro DNA synthess. Large amounts of a specc segment of
DNA, of dened ength and sequence, can be syntheszed from
a sma amount oftempate. PCRs a rapd, senstve and
nexpensve procedure for ampfyng DNA of specc nterest.
The technque has revoutonzed moecuar boogy and s used
n vrtuay every area of natura scences and medcne. The
technque has cut across the boundares separatng basc and
apped research, Commerca technoogy and medcne n a
way few technques ever have.
The prncpa ofPCRs rather smp e and nvoves enzymatc
ampcaton of a DNA fragment anked by two
ogonuceotdes (prmers) hybrdzed to opposte strands of the
tempate wth the 3' ends facng each other (Fgure 1). DNA
poymerase syntheszes new DNA startng from the 3'end of
each prmer. Repeated cyces of heat denaturaton of the
tempate, anneang of the prmers and extenson of the
anneaed prmers by DNApoymerase resuts n ampcaton of
the DNA fragment. The extenson products of each prmer can
serve as tempate for the other prmer resutng n essentay
doubng the amount of the DNA fragment n each cyce. The
resut s an exponenta ncrease n the amount of specc DNA
fragment dened by the 5' ends of the prmers.
The PCRrevouton began quety enough n the mnd ofKary
Mus. By hs own account, Mus, then at Cetus Corporaton,
thought up the technque one Frday nght n the summer of
1983(Mus, 1990). The foowng wnter, havng made some
educated guesses about concentratons and reacton tmes, he
carred out hs rst experment. He was ucky because the rst
tme he tred the reacton, t worked! The nta procedure used
Kenow fragment of E.coliDNApoymerase I added fresh durng
each cyce because the enzyme was nactvated by each
denaturaton step (Sak et a 1985; Mus et a 1986). The
ntroducton of thermostabe DNA poymerase from the
thermophc bacterum Thermus aquaticus (Taq) (Sak et a.
1988) permtted the deveopment of nstruments that
automated the Cycng porton of the procedure, substantay
reducng the work needed to performa PCR.The use of ths
poymerase aso ncreased the speccty and the yed of the
desred product because hgher temperatures for anneang and
extenson coud be used.
Snce ts ntroducton, numerous modcatons and appcatons
of the PCR technque have been deveoped, the presentaton of
even a sma number of them vasty exceeds the scope of ths
book. There are books that are dedcated to ths sub|ect
(Dehenbach and Dvekser 1995; Inns et a 1995; Grmn and
Grmn, 1994; Inns et a, 1990; Whte et a 1980). In ths
chapter, the most basc PCR concepts are presented aong wth
genera protocos. The am s to provde an overa ntroducton
to PCR technoogy that can serve as the bass for
understandng more sophstcated appcatons.
El!n"s o# s"an$ar$ PCR ra%"ion
Components of a standard PCR reacton are: thermostabe DNA
poymerase, DNA tempate, prmers, dNTP substrate, MgCz
buher and sat. In addton, PCR reactons frequenty ncude
compounds that stabze the enzyme and reagents that hep
DNA dssocaton or prmer anneang.
DNA Poymerase
The most commony used thermostabe DNApoymerase s Taq
poymerase. Consequenty most standard PCRprotocos are
optmzed for maksma actvty of ths enzyme. However, Taq
poymerase exhbts some propertes that make t ess than
dea for some appcatons. Frst, the enzyme has very hgh
error rates due to the ack of 3' to 5' exonucease actvty (Sak
et a. 1998). Ths may nterfere wth the preparaton of DNA for
sequencng, as we as wth the nabty to obtan ong
repcaton products. Second, the enzyme adds nuceotdes to 3'
ends n a tempate-ndependent manner, makng the
ampcaton product dmcut to cone. Thrd, the enzyme s
qute expensve.
For these reasons a varety of thermostabe DNApoymerases
from thermophc or hyperthermophc bactera have been
soated, characterzed and commercay ntroduced to PCR.
Tabe 3 presents a st of these poymerases and ther ma|or
characterstcs. Some of these poymerases are used n
standard PCR reactons (e.g., T, Pvo) whe others are used n
sequencng (e.g., Pvo, AmpTherm) or n ong PCR reacton
mxtures (e.g., Tth).
The concentraton of the enzyme n a reacton mxture can
ahect the dety of the product. An excessve amount of
poymerase can resut n ampcaton of nonspecc PCR
products. The recommended amount of enzyme per reacton
for most poymerases s 1 to 5 unts. The most frequenty used
concentraton s 2.0 unts/OO -L1 reacton. However, the
enzyme requrement may vary wth respect to ndvdua
tempate DNA or prmer used. It s aways prudent to use the
enzyme concentraton recommended by the supper snce
enzyme actvty from dherent manufacturer can vary.
DNA Tempate
One of the most mportant features of PCR s that t can be
performed wth very sma quanttes of reatvey mpure
DNA.Even degraded DNAcan be successfuy amped.
Therefore a number of smpe and rapd protocos to purfy
DNAfor PCRhave been deveoped. In ths book some of these
methods are descrbed n chapters 2, 3,4 and 5about
DNApurcaton. In spte of the fact that DNA need not be
absoutey pure, a number of contamnants can decrease the
emcency of ampcaton. The presence of urea, SDS, sodum
acetate and some components euted from agarose can
nterfere wth PCR.Most of these mpurtes can be removed by
washng wth 70%ethano or by reprecptaton of DNA n the
presence of ammonum acetate. Protocos for remova of these
contamnants are descrbed n ths book n the chapters on DNA
purcaton.
Prmers
Genera gudenes for seecton of prmers for PCR are:
Optma prmer set shoud hybrdze to the tempate
emcenty wth neggbe hybrdzaton to other sequences of
the sampe. To assure ths, prmers shoud be at east 20 to 25
bases n ength. However, for some appcatons requrng
mutpe random ntaton (e.g., RAPD anayss), prmers are
usuay 8 to 10 bases n ength.
Prmers, f possbe, shoud have a GCcontent smar to that
of the target. Overa GCcontent of 40%to 60% usuay works
for most reactons. The GC content of both prmer shoud be
smar.
Prmers shoud not have sequences wth sgncant secondary
structure. Therefore, prmer sequence shoud not contan
smpe repeats or pandromc sequences.
Prmer pars shoud not contan compementary sequences to
Beach other. In partcuar prmers wth 3' overaps shoud be
avoded. Ths w reduce the ncdence of "prmer-dmer"
formaton that severey ahects emcency of ampcaton.
Many commerca computer programs exst to desgn prmer
sets. Some exceent freey shared programs are: PRIMER from
Whtehead Insttute for Bomedca Research (http://www-
genome.w.edu) for PC Computer and Ampfy and HyperPCR for
Macntosh computers (ftp://ubo.bo.ndana. edu.)
The PCR reacton requres a moar excess of prmers. Prmer
concentraton s usuay 0.2 to 1 |..M. Usng too much prmer
may resu t n fase ntatons and "prmer-dmer" formaton.
The dstance between prmers can vary consderaby. However,
the emcency of ampcaton decreases consderaby for
dstances greater than 3 kbp. In genera, short fragments
ampfy wth hgher emcency than ong ones. Preferenta
ampcaton of short fragments s probaby due to more
emcent fu-ength extenson by the enzyme n each cyce.
Ampcaton of a very arge fragment s possbe wth
modcatons of standard reacton condtons and by usng a
mxture of DNA poymerases (e.g., Taq and Pfu). These
condtons are referred to as ong PCR. Numerous commerca
kts exst that use propretary reagents to acheve ong PCR.
Substrate
The concentraton of each of dNTP n PCRshoud not exceed
200IlM. Ths amount of substrate s sumcent to synthesze
12.5ug of DNAwhen haf of the nuceotdes are ncorporated, a
quantty far exceedng the amount of DNA syntheszed n
standard PCR (see Tabe 1). The four dNTPs shoud be used at
equvaent concentratons, partcuary when Taq poymerase s
used. Ths w mnmze the error rate of the enzyme. An excess
of nuceotdes nhbts enzyme actvty and can contrbute to
the appearance of fase products. Moreover, when varyng dNTP
concentraton, t shoud be remembered that dNTPs cheate
magnesum on, decreasng ts concentraton n the reacton
mxture.
MgC2 Concentraton
Magnesum on s a requred co-factor for a DNApoymerases.
Moreover magnesum on concentraton may ahect the
foowng:
Prmer anneang.
Temperature of strand dssocaton for tempate and product.
Product speccty.
Formaton of prmer-dmer artfacts and enzyme dety.
Many tempates requre optmzaton of magnesum on
concentraton for emcent, correct ampcaton. The optma
concentratons of Mg++ for each thermophc poymerase are
sted n Tabe 3. They shoud serve as a startng pont for
optmzng the magnesum on concentraton n the reacton.
Buher and Sat
A standard buher for PCR s 10 to 50 mM Trs-HC. The optmum
pH s between 8 and 9 for most thermophc poymerases
(Tabe 3). Snce the L1 pKa for Trs s hgh (- 0.031 jOC), the true
pH of the reacton mxture durng a typca therma cyce vares
consderaby (approxmatey 1 to 1.5 pH unts).
The sat used n most reactons s potassum or sodum. The K+
(Na") s usuay added to factate correct prmer anneang. For
Taq poymerase, the concentraton usuay used s 50mM snce
hgher monovaent on concentratons nhbt poymerase
actvty (Inns et a. 1988).It s not necessary to use sat when
ampfyng DNA usng a rapd cyce ampcaton nstrument,
snce dety of prmer anneang s acheved as a resut of the
short anneang tme. Some other components used n reactons
to hep stabze the enzyme are: geatn, bovne serum abumn
or nononc detergent such as Tween 20or Trton X100.
However, most protocos work we wthout the addton of
these ngredents.
Thr!al %y%lin& 'ro(l
Standard PCR conssts of three steps : denaturaton, anneang
and extenson. These steps are repeated or cyced 25 to 30
tmes. Most protocos aso ncude a snge denaturaton step
(nt a denaturaton) before cycng begns and snge ong
extenson step (na extenson) at the end.
An nta denaturaton that asts for a few mnutes s added to
assure compete denaturaton of the tempate. Ths premnary
step s mportant for compex tempates or tempates wth hgh
GCcontent. The na extenson step s usuay carred out for 5
to 10 mnutes to assure competon of parta extenson
products by DNA poymerase and to provde tme for
compete renaturaton of snge stranded products.
Denaturaton Step
In ths step, compete separaton of the tempate strands must
be accompshed. Denaturaton s a rst order reacton that
occurs very fast. A compete descrpton of the denaturaton
reacton s gven n chapter 10.Astan dard tme 000 to 60
seconds s used to assure unform heatng of the entre reacton
voume. When a rapd cyce nstrument s used, ths tme s
usuay set to 0 for a 10u reacton cyced n a gass capary
tube. The temperatur of cyce denaturaton s usuay 94 C to
95 "C.
Anneang Step
In ths step, prmers annea to the tempate. Wth excess prmer
the anneang reacton s pseudo rst order reacton (see
chapter 10 for an expanaton) and occurs very fast. The tme
used for ths step, n most protocos, s 30 to 60 seconds, the
tme requred to coo the reacton from the denaturaton
temperature to the anneang temperature. In a rapd cyce
nstrument, ths tme s usuay set to O. The anneang
temperature depends on the GC content of the prmers and
shoud be carefuy ad|usted . Lowanneang temperatures w
resut n unspecc prmer anneang and ntaton. Hgh
temperatures w prevent anneang and consequenty w
prevent DNA synthess. For most prmers, an anneang
temperature of 50 C to 55 C s used wthout furthe r
optmzaton.
Eongaton Step
In ths step, DNA poymerase syntheszes a new DNA strand by
extendng the 3' ends of the prmers. Tme of the eongaton
depends on the ength of the sequence to be amped . Snce
Taq poymerase can add 60-100 bases per second under
optma condtons, synthess of a 1 Kbp fragment shoud
requre a tte ess than 20 seconds. However, most protocos
recommend 60 seconds per 1 Kbp DNAto account for tme
needed to reach the correct temperature and to compensate
for other unknown factors that can ahect reacton rate . The
shortest possbe tme shoud be used to preserve poymerase
actvty. In ar rapd cyce nstruments, the eongaton tme s
usuay set to that cacuated for the theoretca eongaton rate
of poymerase (15 to 20 seconds per 1 Kbp of desred
fragment) .
An extenson temperature of 72 C s used n standard
ampcaton protocos. Ths temperature s cose to the optma
temperature for Taq poymerase (75 C) but ow enough to
prevent dssoca ton of the prmer from the tempate. However,
t shoud be remembered that Taq poymerase has substanta
eongaton actvty at 55 C, the temperature used n most
anneang steps . Ths resdua synthess stabzes the
nteracton of prmer wth tempate and ndeed permts the use
of an eongaton temperatur hgher than 72 "C, Hgher
temperatures of eongaton are used when the tempate can
form secondary structures that nterfere wth eongaton.
Prncpes and Technque of Bochemstry and Moecuar Boogy
Wson and Waker
Basc Concept of the PCR
The poymerase chan reacton or PCR s one of the manstays
of moecuar boogy. One of the reasons for the wde adopton
of the PCR s the eegant smpcty of the reacton and reatve
ease of the practca manpuaton steps. Indeed combned wth
the reevant bonformatcs resources for ts desgn and for
determnaton of the requred expermenta condtons t
provdes a rapd means for DNA dentcaton and anayss. It
has opened up the nvestgaton of ceuar and moecuar
processes to those outsde the ed of moecuar boogy.
The PCR s used to ampfy a precse fragment of DNA from a
compex mxture of startng matera usuay termed the
tempate DNA and n many cases requres tte DNA
purcaton. It does requre the knowedge of some DNA
sequence nformaton whch anks the fragment of DNA to be
amped (target DNA). From ths nformaton two
ogonuceotde prmers may be chemcay synthessed each
compementary to a stretch of DNA to the 30 sde of the target
DNA, one ogonuceotde for each of the two DNA strands (Fg.
5.32). It may be thought of as a technque anaogous to the
DNA repcaton process that takes pace n ces snce the
outcome s the same: the generaton of new compementary
DNA stretches based upon the exstng ones. It s aso a
technque that has repaced, n many cases, the tradsona
DNA conng methods snce t fus the same functon, the
producton of arge amounts of DNA from mted startng
matera; however, ths s acheved n a fracton of the tme
needed to cone a DNA fragment (Chapter 6). Athough not
wthout ts drawbacks the PCR s a remarkabe deveopment
whch s changng the approach of many scentsts to the
anayss of nucec acds and contnues to have a profound
mpact on core boscences and botechnoogy.
Stages n PCR
The PCR conssts of three dened sets of tmes and
temperatures termed steps: () denaturaton, () anneang and
() extenson. Each of these steps s repeated 30-40 tmes,
termed cyces (Fg. 5.33). In the rst cyce the doube-stranded
tempate DNA s () denatured by heatng the reacton to above
90 _C. Wthn the compex DNA the regon to be speccay
amped (target) s made accessbe. The temperature s then
cooed to 40-60 _C. The precse temperature s crtca and
each PCR system has to be dened and optmsed. One usefu
technque for optmsaton s touchdown PCR where a
programmabe cycer s used to ncrementay decrease the
anneang temperature unt the optmum s derved. Reactons
that are not optmsed may Gde rse to other DNA products n
addton to the specc target or may not produce any amped
products at a. The anneang step aows the hybrdsaton of
the two ogonuceotde prmers, whch are present n excess, to
bnd to ther compementary stes that ank the target DNA.
The anneaed ogonuceotdes act as prmers for DNA
synthess, snce they provde a free 30 hydroxy group for DNA
poymerase. The DNA
synthess step s termed extenson and s carred out by a
thermostabe DNA poymerase, most commony Taq DNA
poymerase.
DNA synthess proceeds from both of the prmers unt the new
strands have been extended aong and beyond the target DNA
to be amped. It s mportant to note that, snce the new
strands extend beyond the target DNA, they w contan a
regon near ther 30 ends that s compementary to the other
prmer. Thus, f another round of DNA synthess s aowed to
take pace, not ony the orgna strands w be used as
tempates but aso the new strands. Most nterestngy, the
products obtaned from the new strands w have a precse
ength, demted exacty by the two regons compementary to
the prmers. As the system s taken through successve cyces
of denaturaton, anneang and extenson a the new strands
w act as tempates and so there w be an exponenta
ncrease n the amount of DNA produced. The net ehect s to
seectvey ampfy the target DNA and the prmer regons
ankng t (Fg. 5.34).
One probem wth eary PCR reactons was that the temperature
needed to denature the DNA aso denatured the DNA
poymerase. However the avaabty of a thermostabe DNA
poymerase enzyme soated from the thermophc bacterum
Thermus aquatcus found n hot sprngs provded the means to
automate the reacton. Taq DNA poymerase has a temperature
optmum of 72 _C and survves proonged exposure to
temperatures as hgh as 96 _C and so s st actve after each of
the denaturaton steps. The wdespread utty of the technque
s aso due to the abty to automate the reacton and as such
many therma cycers have been produced n whch t s
possbe to program n the temperatures and tmes for a
partcuar PCR reacton.
Moecuar Botechnoogy
Prncpes and Appcatons of Recombnant DNA
Gck, Pasternak, and Patten
Poymerase Chan Reacton
PCR s an ehectve procedure for generatng arge quanttes of
a specc DNA sequence n vtro. Ths ampcaton, whch can
be more than a monfod, s acheved by a three-step cycng
process. The essenta compo nents for PCR ampcaton are
(1) two synthetc ogonuceotde prmer (-20 nuceotdes each)
that are compementary to regon on opposte strands that
ank the target DNA sequence and that, after anneang to the
source DNA, have ther 3 hydroxy ends orented toward each
other; (2) a tempate sequence n a DNA sampe that es
between the prmer-bndng stes and that can be from 100 to
-35,000 bp n ength; (3) a thermostabe DNA poymerase that
can wthstand beng heated to 95C or hgher and that copes
the DNA tempate wth hgh dety; and (4) the four
deoxyrbonuceotdes.
A typca PCR process entas a number of cyces for ampfyng
a specc DNA sequence. Each cyce has three successve
steps.
1. Denaturaton. The rst step n the PCR ampcaton system
s the therma denaturaton of the DNA sampe by rasng the
temperatur wthn a reacton tube to 95C. In addton to the
source tempate DNA, ths reacton tube contans a vast moar
excess of the two ogonuceotde prmers, a thermostabe DNA
poymerase (e.g., Taq DNA poymerase, soated from the
bacterum Thermus aquaticus), and four deoxyrbonuceotdes.
The temperature s mantaned for about 1 mnute.
2. Renaturaton. For the second step, the temperature of the
mxture s sowy owered to -55C. Durng ths step, the
prmers base par wth ther compementary sequences n the
DNA sampe.
3. Synthess. In the thrd step, the temperature s rased to
-75C, whch s optmum for the cataytc functonng of Taq
DNA poymerase. DNA synthess s ntated at the 3 hydroxy
end of each prmer and uses the source DNA as a tempate (Fg.
4.14).
A steps n a PCR cyce are carred out n an automated bock
heater that s programmed to change temperatures after a
speced perod of tme. One cyce generay asts from 3 to 5
mnutes.
To understand how the PCR protoco succeeds n ampfyng a
dscrete segment of DNA, t s mportant to keep n mnd the
ocaton of Beach prmer-anneang ste and ts compementary
sequence wthn the strands that are syntheszed durng each
cyce. Durng the synthess phase of the rst cyce, the newy
syntheszed DNA from each prmer s extended beyond the
endpont of the sequence that s compementary to the second
prmer. These new strands form "ong tempates" that are used
n the second cyce (Fg. 4.14).
Durng the second cyce, the orgna DNA strands and the new
strands syntheszed n the rst cyce (ong tempates) are
denatured and then hybrdzed wth the prmers. The arge
moar excess of prmers n the reacton mxture ensures that
they w hybrdze to the tempate DNA before compementary
tempate strands have the chance to reannea to each other. A
second round of synthess produces ong tempates from the
orgna strands, as we as some DNA strands that have a
prmer sequence at one end and termnate wth a sequence
compementary to the other prmer at the other end ("short
tempates") that were generated from the ong tempates (Fg.
4.15).
Durng the thrd cyce, short tempates, ong tempates, and
orgna strands a hybrdze wth the prmers and are repcated
(Fg. 4.16). In subsequent cyces, the short tempates
preferentay accumuate, and by the 30th cyce, these strands
are about a mon tmes more abundant than ether the
orgna or ong tempate strands (Fg. 4.17). PCR has become a
pervasve technque that s used for nnumerabe purposes,
some of whch are descrbed here and many others n the
ensung chapters.
Gene Conng and DNA Anayss
Chapter 9 The Poymerase Chan Reacton
The PCR outne
The poymerase chan reacton resuts n the seectve ampcaton of a
chosen regon of a DNA moecue. Any regon of any DNA moecue can be
chosen, so ong as the sequences at the borders of the regon are known.
The border sequences must be known because n order to carry out a PCR,
two short ogonuceotdes must hybrdze to the DNA moecue, one to
each strand of the doube hex (Fgure 9.1). These ogonuceotdes, whch
act as prmers for the DNA synthess reactons, demt the regon that w
be amped.
Ampcaton s usuay carred out by the DNA poymerase I enzyme from
Thermus aquaticus. As mentoned on p. 49, ths organsm ves n hot
sprngs, and many of ts enzymes, ncudng Taq poymerase, are
thermostabe, meanng that they are resstan to denaturaton by heat
treatment. As w be apparent n a moment, the thermostabty of Taq
poymerase s an essenta requrement n PCR methodoogy.
To carry out a PCR experment, the target DNA s mxed wth Taq
poymerase, the two ogonuceotde prmers, and a suppy of nuceotdes.
The amount of target DNA can be very sma because PCR s extremey
senstve and w work wth |ust a snget startng moecue. The reacton
s started by heatng the mxture to 94C. At ths temperature the
hydrogen bonds that hod together the two poynuceotdes of the doube
hex are broken, so the target DNA becomes denatured nto snge-
stranded moecues (Fgure 9.2). The temperature s then reduced to 50-
60C, whch resuts n some re|onng of the snge strands of the target
DNA, but aso aows the prmers to attach to ther anneang postons.
DNA synthess can now begn, so the temperature s rased to 74C, |ust
beow the optmum for Taq poymerase. In ths rst stage of the PCR, a set
of "ong products" s syntheszed from each strand of the target DNA.
These poynuceotdes have dentca 5 ends but random 3 ends, the
atter representng postons where DNA synthess termnates by chance.
The cyce of denaturaton-anneang-synthess s now repeated (Fgure
9.3). The ong products denature and the four resutng strands are coped
durng the DNA synthess stage. Ths gves four doube-stranded
moecues, two of whch are dentca to the ong products from the rst
cyce and two of whch are made entrey of new DNA. Durng the thrd
cyce, the atter gve rse to "short products", the 5 and 3 ends of whch
are both set by the prmer anneang postons. In subsequent cyces, the
number of short products accumuates n an exponenta fashon (doubng
durng each cyce) unt one of the components of the reacton becomes
depeted. Ths means that after 30 cyces, there w be over 130 mon
short products derved from each startng moecue. In rea terms, ths
equates to severa mcrograms of PCR product from a few nanograms or
ess of target DNA.
At the end of a PCR a sampe of the reacton mxture s usuay anayzed
by agarose ge eectrophoress, sumcent DNA havng been produced for
the amped fragment to be vsbe as a dscrete band after stanng wth
ethdum bromde. Ths may by tsef provde usefu nformaton about the
DNA regon that has been amped, or aternatvey the PCR product can
be examned by technques such as DNA sequencng.
Desgnng the ogonuceotde prmers for a PCR
The prmers are the key to the success or faure of a PCR experment. If
the prmer are desgned correcty the experment resuts n ampcaton
of a snge DNA fragment, correspondng to the target regon of the
tempate moecue. If the prmers are ncorrecty desgned the experment
w fa, possby because no ampcaton occurs, or possby because the
wrong fragment, or more than one fragment, s amped (Fgure 9.4).
Ceary a great dea of thought must be put nto the desgn of the prmers.
Workng out approprate sequences for the prmers s not a probem: they
must correspond wth the sequences ankng the target regon on the
tempate moecue. Each prmer must, of course, be compementary (not
dentca) to ts tempate strand n order for hybrdzaton to occur, and the
3 ends of the hybrdzed prmers shoud pont toward one another (Fgure
9.5). The DNA fragment to be amped shoud not be greater than about
3 kb n ength and deay ess than 1 kb. Fragments up to 10 kb can be
amped by standard PCR technques, but the onger the fragment the
ess emcent the ampcaton and the more dmcut t s to obtan
consstent resuts. Ampcaton of very ong fragments-up to 40 kb-s
possbe, but requres speca methods.
The rst mportant ssue to address s the ength of the prmers. If the
prmers are too short they mght hybrdze to non-target stes and gve
undesred ampcaton products. To ustrate ths pont, magne that tota
human DNA s used n a PCR experment wth a par of prmers eght
nuceotdes n ength (n PCR |argon, these are caed "8-mers"). The key
resut s that a number of dherent fragments w be amped. Ths s
because attachment stes for these prmers are expected to occur, on
average, once every 48 = 65,536 bp, gvng approxmatey 49,000
possbe stes n the 3,200,000 kb of nuceotde sequence that makes up
the human genome. Ths means that t woud be very unkey that a par
of 8-mer prmers woud gve a snge, specc ampcaton product wth
human DNA (Fgure 9.6a).
What f the 17-mer prmers shown n Fgure 9.5 are used? The expected
frequency of a 17-mer sequence s once every 417 = 17,179,869,184 bp.
Ths gure s over ve tmes greater than the ength of the human
genome, so a 17-mer prmer woud be expected to have |ust one
hybrdzaton ste n tota human DNA. A par of 17-mer prmers shoud
therefore gve a snge, specc ampcaton product (Fgure 9.6b).
Why not smpy make the prmers as ong as possbe? The ength of the
prmer nuences the rate at whch t hybrdzes to the tempate DNA, ong
prmers hybrdzng at a sower rate. The emcency of the PCR, measured
by the number of amped moecues produced durng the experment, s
therefore reduced f the prmers are too ong, as compete hybrdzaton to
the tempate moecues cannot occur n the tme aowed durng the
reacton cyce. In practce, prmers onger than 30-mer are rarey used.
Workng out the correct temperatures to use
Durng each cyce of a PCR, the reacton mxture s transferred between three
temperatures (Fgure 9.7):
The denaturaton temperature, usuay 94C, whch breaks the base pars and
reeases snge-stranded DNA to act as tempates n the next round of DNA
synthess;
The hybrdzaton or anneang temperature, at whch the prmers attach to the
tempates;
The extenson temperature, at whch DNA synthess occurs. Ths s usuay set
at 74C, |ust beow the optmum for Taq poymerase.
The anneang temperature s the mportant one because, agan, ths can ahect
the speccty of the reacton. DNA-DNA hybrdzaton s a temperature-
dependent phenomenon. If the temperature s too hgh no hybrdzaton takes
pace; nstead the prmers and tempates reman dssocated (Fgure 9.8a).
However, f the temperature s too ow, msmatched hybrds-ones n whch not
a the correct base pars have formed-are stabe (Fgure 9.8b). If ths occurs the
earer cacuatons regardng the approprate engths for the prmers become
rreevant, as these cacuatons assumed that ony perfect prmer-tempate
hybrds are abe to form. If msmatches are toerated, the number of potenta
hybrdzaton stes for each prmer s greaty ncreased, and ampcaton s more
key to occur at non-target stes n the tempate moecue.
The dea anneang temperature must be ow enough to enabe hybrdzaton
between prmer and tempate, but hgh enough to prevent msmatched hybrds
from formng (Fgure 9.8c). Ths temperature can be estmated by determnng
the metng temperature or Tm of the prmer-tempate hybrd. The Tm s the
temperature at whch the correcty base-pared hybrd dssocates ("mets"). A
temperature 1-2C beow ths shoud be ow enough to aow the correct prmer-
tempate hybrd to form, but too hgh for a hybrd wth a snge msmatch to be
stabe. The Tm can be determned expermentay but s more usuay cacuated
from the smpe formua (Fgure 9.9):
Tm = (4 |G + C|) + (2 |A + T|)C
n whch |G + C| s the number of G and C nuceotdes n the prmer sequence,
and |A + T| s the number of A and T nuceotdes.
The anneang temperature for a PCR experment s therefore determned by
cacuatng the Tm for each prmer and usng a temperature of 1-2C beow ths
gure. Note that ths means the two prmers shoud be desgned so that they
have dentca Tms. If ths s not the case, the approprate anneang temperature
for one prmer may be too hgh or too ow for the other member of the par.