Contents

1) lnLroducLlon

2) PlsLory


3) polymerase chaln reacLlon(pcr)

4) 8equlres componenL ln pcr



3) procedure


6) deslgn Lhe prlmer

7) prlmer exLenslon


8) appllcaLlon of pcr

9) absLracL


10) recenL dlscovery










9o|ymerase cha|n react|on
lnLroducLlon and hlsLory of pcr
C8 (olymerase Chaln 8eacLlon) ls a revoluLlonary meLhod developed by kary Mullls ln Lhe 1980s C8
ls based on uslng Lhe ablllLy of unA polymerase Lo synLheslze new sLrand of unA complemenLary Lo Lhe
offered LemplaLe sLrand 8ecause unA polymerase can add a nucleoLlde only onLo a preexlsLlng 3CP
group lL needs a prlmer Lo whlch lL can add Lhe flrsL nucleoLlde 1hls requlremenL makes lL posslble Lo
dellneaLe a speclflc reglon of LemplaLe sequence LhaL Lhe researcher wanLs Lo ampllfy AL Lhe end of Lhe
C8 reacLlon Lhe speclflc sequence wlll be accumulaLed ln bllllons of coples (ampllcons)



Principle of the PCR


1he purpose of a C8 (olymerase Chaln 8eacLlon) ls Lo make a huge number of
coples of a gene 1hls ls necessary Lo have enough sLarLlng LemplaLe for sequenclng
@he cyc||ng react|ons
1here are Lhree ma[or sLeps ln a C8 whlch are repeaLed for 30 or 40 cycles
1hls ls done on an auLomaLed cycler whlch can heaL and cool Lhe Lubes wlLh
Lhe reacLlon mlxLure ln a very shorL Llme
1 enaturat|on aL 94C

uurlng Lhe denaLuraLlon Lhe double sLrand melLs open Lo slngle
sLranded unA all enzymaLlc reacLlons sLop (for example Lhe exLenslon
from a prevlous cycle)
2 nnea||ng aL 34C

1he prlmers are [lggllng around caused by Lhe 8rownlan moLlon lonlc
bonds are consLanLly formed and broken beLween Lhe slngle sLranded
prlmer and Lhe slngle sLranded LemplaLe 1he more sLable bonds lasL a
llLLle blL longer (prlmers LhaL flL exacLly) and on LhaL llLLle plece of
double sLranded unA (LemplaLe and prlmer) Lhe polymerase can aLLach
and sLarLs copylng Lhe LemplaLe Cnce Lhere are a few bases bullL ln
Lhe lonlc bond ls so sLrong beLween Lhe LemplaLe and Lhe prlmer LhaL lL
does noL break anymore
3 extens|on aL 72C

1hls ls Lhe ldeal worklng LemperaLure for Lhe polymerase 1he prlmers
where Lhere are a few bases bullL ln already have a sLronger lonlc
aLLracLlon Lo Lhe LemplaLe Lhan Lhe forces breaklng Lhese aLLracLlons
rlmers LhaL are on poslLlons wlLh no exacL maLch geL loose agaln
(because of Lhe hlgher LemperaLure) and donL glve an exLenslon of Lhe
fragmenL
1he bases (complemenLary Lo Lhe LemplaLe) are coupled Lo Lhe prlmer
on Lhe 3 slde (Lhe polymerase adds dn1s from 3 Lo 3 readlng Lhe
LemplaLe from 3 Lo 3 slde bases are added complemenLary Lo Lhe
LemplaLe)


llgure 3 1he dlfferenL sLeps ln C8 (pdf flle of Lhls plcLure)
AnlmaLed plcLure of C8
8ecause boLh sLrands are copled durlng C8 Lhere ls an exponent|a| lncrease
of Lhe number of coples of Lhe gene Suppose Lhere ls only one copy of Lhe
wanLed gene before Lhe cycllng sLarLs afLer one cycle Lhere wlll be 2 coples
afLer Lwo cycles Lhere wlll be 4 coples Lhree cycles wlll resulL ln 8 coples and
so on


llgure 4 1he exponenLlal ampllflcaLlon of Lhe gene ln C8
s there a gene cop|ed dur|ng 9Ck and |s |t the r|ght s|ze ?
8efore Lhe C8 producL ls used ln furLher appllcaLlons lL has Lo be checked lf
1 1here ls a producL formed
1hough blochemlsLry ls an exacL sclence noL every C8 ls successful
1here ls for example a posslblllLy LhaL Lhe quallLy of Lhe unA ls poor
LhaL one of Lhe prlmers doesnL flL or LhaL Lhere ls Loo much sLarLlng
LemplaLe
2 1he producL ls of Lhe rlghL slze
lL ls posslble LhaL Lhere ls a producL for example a band of 300 bases
buL Lhe expecLed gene should be 1800 bases long ln LhaL case one of
Lhe prlmers probably flLs on a parL of Lhe gene closer Lo Lhe oLher
prlmer lL ls also posslble LhaL boLh prlmers flL on a LoLally dlfferenL
gene
3 Cnly one band ls formed
As ln Lhe descrlpLlon above lL ls posslble LhaL Lhe prlmers flL on Lhe
deslred locaLlons and also on oLher locaLlons ln LhaL case you can
have dlfferenL bands ln one lane on a gel






llgure 3 verlflcaLlon of Lhe C8 producL on gel
1he ladder ls a mlxLure of fragmenLs wlLh known slze Lo compare wlLh Lhe C8
fragmenLs noLlce LhaL Lhe dlsLance beLween Lhe dlfferenL fragmenLs of Lhe
ladder ls logarlLhmlc Lane 1 C8 fragmenL ls approxlmaLely 1830 bases long
Lane 2 and 4 Lhe fragmenLs are approxlmaLely 800 bases long Lane 3 no
producL ls formed so Lhe C8 falled Lane 3 mulLlple bands are formed
because one of Lhe prlmers flLs on dlfferenL places







Polymerase chain reaction
"PCR" redirects here. For other uses, see PCR (disambiguation).


A strip of eight PCR tubes, each containing a 100 l reaction mixture
The poIymerase chain reaction (PCR) is a scientific technique in molecular biologyto amplify a single or a few
copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence.
Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and
biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-
based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic
fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.
n 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.
The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA
melting and enzymatic replication of the DNA. Primers(short DNA fragments) containing sequences complementary
to the target region along with a DNA polymerase (after which the method is named) are key components to enable
selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for
replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be
extensively modified to perform a wide array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally
isolated from the bacterium%hermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand
from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA
oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of
PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of
temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA
double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used
as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of
PCR results from the use of primers that are complementary to the DNA region targeted for amplification under
specific thermal cycling conditions.




@he 9Ck react|on requ|res the fo||ow|ng components
unA LemplaLe Lhe sample unA LhaL conLalns Lhe LargeL sequence AL Lhe beglnnlng of Lhe reacLlon
hlgh LemperaLure ls applled Lo Lhe orlglnal doublesLranded unA molecule Lo separaLe Lhe sLrands from
each oLher
unA polymerase a Lype of enzyme LhaL synLheslzes new sLrands of unA complemenLary Lo Lhe LargeL
sequence 1he flrsL and mosL commonly used of Lhese enzymes ls 1aq unA polymerase (from 1hermls
aquaLlcus) whereas fu unA polymerase (from yrococcusfurlosus) ls used wldely because of lLs hlgher
fldellLy when copylng unA AlLhough Lhese enzymes are subLly dlfferenL Lhey boLh have Lwo capablllLles
LhaL make Lhem sulLable for C8 1) Lhey can generaLe new sLrands of unA uslng a unA LemplaLe and
prlmers and 2) Lhey are heaL reslsLanL

rlmers shorL pleces of slnglesLranded unA LhaL are complemenLary Lo Lhe LargeL sequence 1he
polymerase beglns synLheslzlng new unA from Lhe end of Lhe prlmer

nucleoLldes (dn1s or deoxynucleoLlde LrlphosphaLes) slngle unlLs of Lhe bases A 1 C and C whlch
are essenLlally bulldlng blocks for new unA sLrands


81C8 (8everse 1ranscrlpLlon C8) ls C8 preceded wlLh converslon of sample 8nA lnLo cunA wlLh
enzyme reverse LranscrlpLase






PCR principles and procedure


igure 1a A thermal cycler for PCR

igure 1b An older model three-temperature thermal cycler for PCR
PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA
fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb
in size.
A basic PCR set up requires several components and reagentsThese components include
template that contains the DNA region (target) to be amplified.
Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sensestrand of
the DNA target.
%aq polymerase or another DNA polymerase with a temperature optimum at around 70 C.
eoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups), the building-blocks from
which the DNA polymerase synthesizes a new DNA strand.
uffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA
polymerase.
ivalent cations, magnesium or manganese ions; generally Mg
2+
is used, but Mn
2+
can be utilized for PCR-
mediated DNA mutagenesis, as higher Mn
2+
concentration increases the error rate during DNA synthesis
onovalent cation potassium ions.
The PCR is commonly carried out in a reaction volume of 10200 l in small reaction tubes (0.20.5 ml volumes) in
a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each
step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect, which permits both
heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction
tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated
lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of
oil on top of the reaction mixture or a ball of wax inside the tube.
Procedure


igure 2 Schematic drawing of the PCR cycle. (1) Denaturing at 94-96 C. (2) AnneaIing at ~65 C (3) EIongation at 72 C. Four cycles
are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase
(light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.
Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly
consisting of 2-3 discrete temperature steps, usually three (Fig. 2). The cycling is often preceded by a single
temperature step (called hold) at a high temperature (>90C), and followed by one hold at the end for final product
extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a
variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and
dNTPs in the reaction, and the melting temperature (Tm) of the primers.
nitialization step This step consists of heating the reaction to a temperature of 9496 C (or 98 C if extremely
thermostable polymerases are used), which is held for 19 minutes. t is only required for DNA polymerases that
require heat activation byhot-start PCR.
enaturation step This step is the first regular cycling event and consists of heating the reaction to 9498 C for
2030 seconds. t causes DNA melting of the DNA template by disrupting the hydrogen bonds between
complementary bases, yielding single-stranded DNA molecules.
nnealing step The reaction temperature is lowered to 5065 C for 2040 seconds allowing annealing of the
primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius
below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence
very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins
DNA synthesis.
xtension/elongation step The temperature at this step depends on the DNA polymerase used; Taq
polymerase has its optimum activity temperature at 7580 C,
[10][11]
and commonly a temperature of 72 C is
used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the
DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing
the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA
strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment
to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand
bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or
reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric)
amplification of the specific DNA fragment.
Final elongation This single step is occasionally performed at a temperature of 7074 C for 515 minutes after
the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
Final hold This step at 415 C for an indefinite time may be employed for short-term storage of the reaction.


igure 3 Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence
from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful
amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for
sizing the bands in the experimental PCRs.
To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer
or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR
products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA
fragments of known size, run on the gel alongside the PCR products

PCR stages
The PCR process can be divided into three stages
xponential amplification At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The
reaction is very sensitive only minute quantities of DNA need to be present.
Leveling off stage The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as
dNTPs and primers causes them to become limiting.
Plateau No more product accumulates due to exhaustion of reagents and enzyme.
PCR optimization
n practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of
spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing
PCR conditions. Contamination with extraneous DNA is addressed with lab protocols and procedures that separate
pre-PCR mixtures from potential DNA contaminants.This usually involves spatial separation of PCR-setup areas from
areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning the work
surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in
avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes
can help with amplification of long or otherwise problematic regions of DNA. Addition of reagents, such as formamide,
in buffer systems may increase the specificity and yield of PCR
Pr|mer 0es|gn

Researcrers agreed ear|y or lral lre des|gr ol PCR pr|rers Was d|ll|cu|l ard urre||ao|e. Corpuler progrars Were dev|sed lo la|e a|| ol lre
des|gr cr|ler|a |rlo accourl. 0re ol lre l|rsl progrars Wr|ller lor pr|rer des|gr Was 0|ga Wr|cr rade use ol lre |rp|ererlal|or ol 0|g|la|
Researcr 0EV (0rapr|cs Erv|rorrerl Varager) or lre Alar| 3T (29). 0|ga Was spec|l|ca||y su|led lo lre po|yrerase cra|r reacl|or (PCR)
a||oW|rg s|ru|lareous ara|ys|s ol lWo pr|rer sequerces. Tre advarlage ol 0|ga Was lral |l prov|ded |r ore prograr ara|yses lor d|recl
repeals, secordary slruclures ard pr|rer d|rer|zal|or as We|| as severa| uselu| 'l|r|sr|rg' loo|s lor Wor|ers ergaged |r PCR opl|r|zal|or ard
o||goruc|eol|de syrlreses. Tre Pr|rer3 prograr al lre wr|leread lrsl|lule |s roW lrougrl lo oe lre rosl re||ao|e ard versal||e loo| currerl|y
ava||ao|e (30).

pp||cat|ons of 9Ck


clonlng geneLlc englneerlng sequenclng

C8 Lechnology has become an essenLlal research and dlagnosLlc Lool for lmprovlng human healLh and
quallLy of llfe C8 Lechnology allows sclenLlsLs Lo Lake a speclmen of geneLlc maLerlal even from [usL
one cell copy lLs geneLlc sequence over and over and generaLe a LesL sample sufflclenL Lo deLecL Lhe
presence or absence of a speclflc vlrus bacLerlum or any parLlcular sequence of geneLlc maLerlal
Medlcal research and cllnlcal medlclne are beneflLlng from C8 Lechnology malnly ln Lwo areas

ueLecLlon of lnfecLlous organlsms lncludlng Lhe vlruses LhaL cause AluS and hepaLlLls and oLher
mlcroorganlsms LhaL affecL womens healLh and cause Luberculosls
ueLecLlon of geneLlc varlaLlons lncludlng muLaLlons ln human genes
9Ck and nfect|ous |sease
C8 Lechnology faclllLaLes Lhe deLecLlon of unA or 8nA of paLhogenlc organlsms and as such ls Lhe
basls for a broad range of cllnlcal dlagnosLlc LesLs for varlous lnfecLlous agenLs lncludlng vlruses and
bacLerla 1hese C8based LesLs have several advanLages over LradlLlonal anLlbodybased dlagnosLlc
meLhods LhaL measure Lhe bodys lmmune response Lo a paLhogen ln parLlcular C8based LesLs are
able Lo deLecL Lhe presence of paLhogenlc agenLs earller Lhan serologlcallybased meLhods as paLlenLs
can Lake weeks Lo develop anLlbodles agalnsL an lnfecLlous agenL Larller deLecLlon of lnfecLlon can
mean earller LreaLmenL and an earller reLurn Lo good healLh

CaplLallzlng on lLs exqulslLe senslLlvlLy sclenLlsLs have also developed C8based LesLs deslgned Lo
quanLlfy Lhe amounL of vlrus ln a persons blood (vlral load) Lhereby allowlng physlclans Lo monlLor
Lhelr paLlenLs dlsease progresslon and response Lo Lherapy vlral load assessmenL before durlng and
afLer Lherapy has Lremendous poLenLlal for lmprovlng Lhe cllnlcal managemenL of dlseases caused by
vlral lnfecLlon lncludlng AluS and hepaLlLls

C8based dlagnosLlcs LesLs are avallable for deLecLlng and/or quanLlfylng several paLhogens lncludlng

Plv1 whlch causes AluS
PepaLlLls 8 and C vlruses whlch can lead Lo llver cancer
Puman aplllomavlrus whlch can cause cervlcal cancer
Chlamydla LrachomaLls whlch can lead Lo lnferLlllLy ln women
nelsserla gonorrhoeae whlch can lead Lo pelvlc lnflammaLory dlsease ln women
CyLomegalovlrus whlch can cause llfe LhreaLenlng dlsease ln LransplanL paLlenLs and oLher
lmmunocompromlsed people lncludlng Plv1/AluS paLlenLs
MycobacLerlum Luberculosls whlch ln lLs acLlve sLaLe causes cough and faLlgue and can lead Lo Llssue
damage of lnfecLed organs
9Ck and 8|ood Screen|ng

Slnce Lhe 1970s serologlcal LesLs have been used Lo screen donaLed blood samples for Lhe presence of
lnfecLlous agenLs neverLheless a small rlsk of vlral Lransmlsslon remalns prlmarlly due Lo Lhe fallure of
such screenlng LesLs Lo ldenLlfy recenLly lnfecLed donors durlng Lhe wlndow perlod Lhe Llme delay
posLlnfecLlon ln whlch Lhe body develops an lmmune response Lo Lhe lnfecLlous agenL 1esLs uslng C8
nuclelc acld ampllflcaLlon LesLlng (nA1) Lechnology deLecL Lhe acLual vlrus LxperLs belleve LhaL lncludlng
such LesLs ln blood screenlng programs could provlde an added measure of proLecLlon by deLecLlng vlral
lnfecLlon aL an earller sLage

Plghly senslLlve C8based LesLs are avallable for deLecLlng early markers of Plv1 hepaLlLls 8 and
hepaLlLls C lnfecLlon namely vlral unA or 8nA ln blood 1hese LesLs are helplng Lo narrow Lhe wlndow
perlod resulLlng ln lmproved blood safeLy 8eneflLs of uslng C8 Lechnology Lo monlLor Lhe worlds
blood supply lnclude

A decrease ln Lhe walLlng perlod or wlndow perlod durlng whlch Lhe lnfecLlous agenL ls undeLecLable
by LradlLlonal screenlng Lechnologles LhaL rely on Lhe formaLlon of anLlbodles
1he ablllLy Lo perform comprehenslve and comblned blood screenlng for several paLhogens lncludlng
hepaLlLls C hepaLlLls 8 and Plv1 ComblnaLlon C8based LesLs lncorporaLed lnLo blood donor screenlng
programs could dramaLlcally reduce Lhe rlsk of vlrus Lransmlsslon1
18lood SafeLy ln Lhe new Mlllennlum publlshed by Lhe Amerlcan AssoclaLlon of 8lood 8anks 2001

9Ck and Genet|c @est|ng

8ecause C8 Lechnology can be used Lo easlly dlsLlngulsh among Lhe Llny varlaLlons ln unA LhaL make
people geneLlcally unlque Lhe Lechnology ls leadlng Lo new meLhods for geneLlc LesLlng Avallable Loday
for dlagnoslng a handful of dlsorders lncludlng CysLlc llbrosls ln Lhe fuLure C8 Lechnology may be used
ln predlcLlve LesLsmeLhods for flndlng ouL who ls predlsposed Lo common dlsorders such as hearL
dlsease and many cancers

8oche ls developlng new molecularbased LesLs ln dlsease predlsposlLlon cancer screenlng and cancer
Lherapy selecLlon and pharmacogeneLlcs a process LhaL deLermlnes how a person responds Lo a drug
based on hls or her geneLlc makeup CeneLlc analysls can provlde lnformaLlon as Lo wheLher an
lndlvldual has Lhe correcL meLabollc paLhway Lo meLabollze a parLlcular drug or even produces Lhe
correcL geneLlc LargeL for Lhe drug As a resulL physlclans wlll noL have Lo rely on Lrlal and error when
prescrlblng Lheraples Lo paLlenLs lnsLead docLors wlll have a clearer undersLandlng of whlch LreaLmenLs
wlll work besL for paLlenLs conLrlbuLlng Lo beLLer Lheraples a subsLanLlal savlngs ln Lhe cosL of Medlcal
appllcaLlons

C8 has been applled Lo a large number of medlcal procedures
1he flrsL appllcaLlon of C81 was for geneLlc LesLlng where a sample of unA ls analyzed for Lhe
presence of geneLlc dlsease muLaLlons rospecLlve parenLs can be LesLed for belng geneLlc carrlers or
Lhelr chlldren mlghL be LesLed for acLually belng affecLed by a dlsease unA samples for renaLal LesLlng
can be obLalned by amnlocenLesls chorlonlc vlllus sampllng or even by Lhe analysls of rare feLal cells
clrculaLlng ln Lhe moLhers bloodsLream C8 analysls ls also essenLlal Lo relmplanLaLlon geneLlc
dlagnosls where lndlvldual cells of a developlng embryo are LesLed for muLaLlons
C8 can also be used as parL of a senslLlve LesL for Llssue Lyplng vlLal Lo organ LransplanLaLlon As of
2008 Lhere ls even a proposal Lo replace Lhe LradlLlonal anLlbodybased LesLs for blood Lype wlLh C8
based LesLs
Many forms of cancer lnvolve alLeraLlons Lo oncogenes 8y uslng C8based LesLs Lo sLudy Lhese
muLaLlons Lherapy reglmens can someLlmes be lndlvldually cusLomlzed Lo a paLlenL
nfect|ous d|sease app||cat|ons

CharacLerlzaLlon and deLecLlon of lnfecLlous dlsease organlsms have been revoluLlonlzed by C8
1he Puman lmmunodeflclency vlrus (or Plv) responslble for AluS ls a dlfflculL LargeL Lo flnd and
eradlcaLe 1he earllesL LesLs for lnfecLlon relled on Lhe presence of anLlbodles Lo Lhe vlrus clrculaLlng ln
Lhe bloodsLream Powever anLlbodles donL appear unLll many weeks afLer lnfecLlon maLernal
anLlbodles mask Lhe lnfecLlon of a newborn and LherapeuLlc agenLs Lo flghL Lhe lnfecLlon donL affecL
Lhe anLlbodles C8 LesLs have been developed LhaL can deLecL as llLLle as one vlral genome among Lhe
unA of over 30000 hosL cells3 lnfecLlons can be deLecLed earller donaLed blood can be screened
dlrecLly for Lhe vlrus newborns can be lmmedlaLely LesLed for lnfecLlon and Lhe effecLs of anLlvlral
LreaLmenLs can be quanLlfled
Some dlsease organlsms such as LhaL for 1uberculosls are dlfflculL Lo sample from paLlenLs and slow Lo
be grown ln Lhe laboraLory C8based LesLs have allowed deLecLlon of small numbers of dlsease
organlsms (boLh llve or dead) ln convenlenL samples ueLalled geneLlc analysls can also be used Lo
deLecL anLlbloLlc reslsLance allowlng lmmedlaLe and effecLlve Lherapy 1he effecLs of Lherapy can also
be lmmedlaLely evaluaLed
1he spread of a dlsease organlsm Lhrough populaLlons of domesLlc or wlld anlmals can be monlLored by
C8 LesLlng ln many cases Lhe appearance of new vlrulenL subLypes can be deLecLed and monlLored
1he subLypes of an organlsm LhaL were responslble for earller epldemlcs can also be deLermlned by C8
analysls
Iorens|c app||cat|ons

1he developmenL of C8based geneLlc (or unA) flngerprlnLlng proLocols has seen wldespread
appllcaLlon ln forenslcs
ln lLs mosL dlscrlmlnaLlng form CeneLlc flngerprlnLlng can unlquely dlscrlmlnaLe any one person from
Lhe enLlre populaLlon of Lhe world MlnuLe samples of unA can be lsolaLed from a crlme scene and
compared Lo LhaL from suspecLs or from a unA daLabase of earller evldence or convlcLs Slmpler
verslons of Lhese LesLs are ofLen used Lo rapldly rule ouL suspecLs durlng a crlmlnal lnvesLlgaLlon
Lvldence from decadesold crlmes can be LesLed conflrmlng or exoneraLlng Lhe people orlglnally
convlcLed
Less dlscrlmlnaLlng forms of unA flngerprlnLlng can help ln arenLal LesLlng where an lndlvldual ls
maLched wlLh Lhelr close relaLlves unA from unldenLlfled human remalns can be LesLed and compared
wlLh LhaL from posslble parenLs slbllngs or chlldren Slmllar LesLlng can be used Lo conflrm Lhe
blologlcal parenLs of an adopLed (or kldnapped) chlld 1he acLual blologlcal faLher of a newborn can also
be conflrmed (or ruled ouL)


kesearch app||cat|on

C8 has been applled Lo many areas of research ln molecular geneLlcs
C8 allows rapld producLlon of shorL pleces of unA even when noLhlng more Lhan Lhe sequence of Lhe
Lwo prlmers ls known 1hls ablllLy of C8 augmenLs many meLhods such as generaLlng hybrldlzaLlon
probes for SouLhern or norLhern bloL hybrldlzaLlon C8 supplles Lhese Lechnlques wlLh large amounLs of
pure unA someLlmes as a slngle sLrand enabllng analysls even from very small amounLs of sLarLlng
maLerlal
1he Lask of unA sequenclng can also be asslsLed by C8 known segmenLs of unA can easlly be
produced from a paLlenL wlLh a geneLlc dlsease muLaLlon ModlflcaLlons Lo Lhe ampllflcaLlon Lechnlque
can exLracL segmenLs from a compleLely unknown genome or can generaLe [usL a slngle sLrand of an
area of lnLeresL
C8 has numerous appllcaLlons Lo Lhe more LradlLlonal process of unA clonlng lL can exLracL segmenLs
for lnserLlon lnLo a vecLor from a larger genome whlch may be only avallable ln small quanLlLles uslng a
slngle seL of vecLor prlmers lL can also analyze or exLracL fragmenLs LhaL have already been lnserLed
lnLo vecLors Some alLeraLlons Lo Lhe C8 proLocol can generaLe muLaLlons (general or slLedlrecLed) of
an lnserLed fragmenL
SequenceLagged slLes ls a process where C8 ls used as an lndlcaLor LhaL a parLlcular segmenL of a
genome ls presenL ln a parLlcular clone 1he Puman Cenome ro[ecL found Lhls appllcaLlon vlLal Lo
mapplng Lhe cosmld clones Lhey were sequenclng and Lo coordlnaLlng Lhe resulLs from dlfferenL
laboraLorles
An exclLlng appllcaLlon of C8 ls Lhe phylogenlc analysls of unA from anclenL sources such as LhaL found
ln Lhe recovered bones of neanderLhals or from frozen Llssues of MammoLhs ln some cases Lhe hlghly
degraded unA from Lhese sources mlghL be reassembled durlng Lhe early sLages of ampllflcaLlon
A common appllcaLlon of C8 ls Lhe sLudy of paLLerns of gene expresslon 1lssues (or even lndlvldual
cells) can be analyzed aL dlfferenL sLages Lo see whlch genes have become acLlve or whlch have been
swlLched off 1hls appllcaLlon can also use CC8 Lo quanLlLaLe Lhe acLual levels of expresslon
1he ablllLy of C8 Lo slmulLaneously ampllfy several locl from lndlvldual sperm4 has greaLly enhanced
Lhe more LradlLlonal Lask of geneLlc mapplng by sLudylng chromosomal crossovers afLer melosls 8are
crossover evenLs beLween very close locl have been dlrecLly observed by analyzlng Lhousands of
lndlvldual sperms Slmllarly unusual deleLlons lnserLlons LranslocaLlons or lnverslons can be analyzed
all wlLhouL havlng Lo walL (or pay for) Lhe long and laborlous processes of ferLlllzaLlon embryogenesls
eLc
thers

C8 ls also lmporLanL ln answerlng baslc sclenLlflc quesLlons ln Lhe fleld of evoluLlonary blology C8 has
been used Lo esLabllsh relaLlonshlps among specles ln anLhropology lL has used Lo undersLand anclenL
human mlgraLlon paLLerns ln archaeology lL has been used Lo help ldenLlfy anclenL human remalns
aleonLologlsLs have used C8 Lo ampllfy unA from exLlncL lnsecLs preserved ln amber for 20 mllllon
years 1he Puman Cenome ro[ecL whlch had a goal of deLermlnlng Lhe sequence of Lhe 3 bllllon base
palrs ln Lhe human genome relled heavlly on C8 1he genes responslble for a varleLy of human
dlseases have been ldenLlfled uslng C8 lor example a C8 Lechnlque called mulLlplex C8 ldenLlfles a
muLaLlon ln a gene ln boys sufferlng from uuchenne muscular dysLrophy C8 can also be used Lo search
for unA from forelgn organlsms such as vlruses or bacLerla
L|m|tat|ons of 9Ck and k@9Ck

1he C8 reacLlon sLarLs Lo generaLe coples of Lhe LargeL sequence exponenLlally Cnly durlng Lhe
exponenLlal phase of Lhe C8 reacLlon ls lL posslble Lo exLrapolaLe back Lo deLermlne Lhe sLarLlng
quanLlLy of Lhe LargeL sequence conLalned ln Lhe sample 8ecause of lnhlblLors of Lhe polymerase
reacLlon found ln Lhe sample reagenL llmlLaLlon accumulaLlon of pyrophosphaLe molecules and self
anneallng of Lhe accumulaLlng producL Lhe C8 reacLlon evenLually ceases Lo ampllfy LargeL sequence aL
an exponenLlal raLe and a plaLeau effecL occurs maklng Lhe end polnL quanLlflcaLlon of C8 producLs
unrellable 1hls ls Lhe aLLrlbuLe of C8 LhaL makes 8eal1lme CuanLlLaLlve 81C8 so neces
4nc|us|4ns

ll |s sa|d lre s|rp|esl ard rosl corver|erl Way lo del|re PCR |s as a 90.36:0. loWever, sucr a calegor|zal|or
e||r|rales lre r|slory ol PCR's deve|oprerl as rary |rd|v|dua|s over lre years corlr|ouled lo lre |deas oer|rd lre
lreory ol PCR ard lre l|re-lur|rg ol lre lecrr|que. Tre rexl s|rp|esl arsWer |s lo rare ar |rd|v|dua| as lre
|rverlor ol lre po|yrerase cra|r reacl|or. Karry Vu|||s Was aWarded lre Nooe| Pr|ze lor Crer|slry |r 1993 lor r|s
d|scovery ol PCR. loWever, lr|s d|scovery |s corlesled arorgsl rary sc|erl|sls, a|| ol Wr|cr ray rave corlr|ouled
lo ur|oc||rg lr|s puzz|e.
ll ras a|so oeer sa|d lral PCR d|d rol ex|sl url|| |l Was rade lo Wor| |r ar exper|rerla| sysler. w|lr lr|s |r r|rd,
rere|y lre lrougrl ol a corcepl |s rol sull|c|erl; a corcepl rusl rave oeer successlu||y oeer pul |rlo pracl|ce (33).
A|lrougr lrere |s douol as lo lre u|l|rale crealor ol PCR, ard douol as lo lre poss|o|||ly lral PCR ray soreroW or
sorel|re oe rep|aced, lrere |s ||ll|e douol lre |rpacl lral PCR ras crealed over a srorl l|re spar or lre sludy ol
ro|ecu|ar oo||




References
1. ) Saiki RK et al. "Enzymatic Amplification of -globin Genomic Sequences and Restriction Site Analysis for
Diagnosis of Sickle Cell Anemia" Science vol. 230 pp. 1350-54 (1985).
2. ) Quill E "Blood-Matching Goes Genetic" Science Magazine (14 March 2008) pp. 1478-1479.
3. ) Kwok S et al. "dentification of HV sequences by using in vitro enzymatic amplification and oligomer
cleavage detection." J. Virol. vol. 61(5) pp. 1690-4 (1987).
4. ) Boehnke M et al. "Fine-structure genetic mapping of human chromosomes using the polymerase chain
reaction on single sperm." Am J Hum Genet vol. 45(1) pp. 21-32 (1989)













$%#%


The polymerase chain reaction (PCR) is a laboratory technique for
"amplifying" a specific DNA sequence. PCR is extremely efficient
and sensitive; it can make millions or billions of copies of any
specific sequence of DNA, even when the sequence is in a
complex mixture. Because of this power, researchers can use it
to amplifysequences even if they only have a minute amount of
DNA. A single hair root, or a microscopic blood stain left at a
crime scene, for example, contains ample DNA for PCR.
PCR has revolutionized the field of molecular biology. It has
enabled researchers to perform experiments easily that
previously had been unthinkable. Before the mid-1980s, when
PCR was developed, molecular biologists had to use laborious and
time-consuming methods to identify, clone, and purify DNA
sequences they wanted to study. Kary Mullis was awarded the
1993 Nobel Prize in Chemistry for inventing PCR.
PCR is based on the way cells replicate their DNA. During DNA
replication, the two strands of each DNA molecule separate, and
DNA polymerase, an enzyme, assembles nucleotides to form two
new partner strandsfor each of the original strands. The original
strands serve as templates for the new strands. The new
strands are assembled such that each nucleotide in the new
strand is determined by the corresponding nucleotide in the
template strands.