Lecture 9 PCR

7/23/2019 Lecture 9 PCR

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Lecture 9:Polymerase Chain Reaction

(PCR)


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Overview

PCR: is a molecular biology technique to amplify ashort region of a DNA molecule by million fold ormore

PCR can be used to clone a given DNA sequence invitrowithout the use of living cells during thecloning process

Developers: Kary Mullis who received a NobelPri!e in "##$ for this wor% invented the method in"#&$


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'he Nobel Pri!e in Chemistry "##$ was awarded "for contributions to the

developments of methods within DNA-based chemistry


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Polymerase Chain ReactionThe PCR method acopying machine for DNAmoleculesDNA molecules can be mass(produced from incredibly small

amounts of material with PCR)Kary Mullis* discovery allowsthe chemist to mimic the cell*sown natural DNA replicationprocess in a test tube) +t hasnow become much easier to

characterise and compare thegenetic material from differentindividuals and organisms)

The Nobel Prize in Chemistry 199


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Princi!les o" PCR

PCR uses synthetic oligonucleotidescomplementary to %nown sequences to primeen!ymatic amplification of the intervening

segment of DNA in the test tube

Carried out by the DNA polymerase + en!yme,normally from Thermus aquaticus-

'his organism lives in hot springs and many ofits en!ymes including the Taqpolymerase are

thermostable


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A repetitive series of cycles involving template

denaturation primers annealing and theextensionof the annealed primers by DNApolymerase results in the exponentialaccumulation of a specific fragment

Primer e.tension products synthesi!ed in onecycle serve as a template in the ne.t cycles thus

/0 cycles of PCR yields about a million(fold ,//0

-amplification


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The Proce#ure

1ost PCR methods typically amplify DNAfragments from few hundred bp up to 2"0 %b

A basic PCR set up requires several componentsand reagentsin a reaction volume of "03/004l insmall reaction tubes ,0)/30)5 ml volumes-

'he reaction is set up in a thin walled PCR tubepermit favorable thermal conductivity to allow forrapid thermal equilibration in a thermal cycler


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The Com!onents o" a PCR Reaction

DNA template that contains the DNA region ,target-to be amplified) 'emplate DNA containing targetsequences can be added to PCR in single( or double(stranded form) PCR requires only a single copy of a

target sequence as template

'wo syntheticoligonucleotide primersthat arecomplimentary to regions on opposite strands that flan%

the target DNA sequence

A thermostable DNA polymerase that can withstandheating #5oC or higher and to catalyse template(dependent

synthesis of DNA


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'he four deo.yribonucleotides ,dNTPs- the building(bloc%sfrom which the DNA polymerase synthesi!es a new DNA strand)6tandard PCR contains equimolar amounts of dA'P d''PdC'P and d7'P ,e)g) /0(/00 41 each depend on e.periment-)8igh concentrations of dN'Ps ,9 m1- are inhibitory becauseof sequestering of 1g/; ion

Buffer solution providing a suitable chemical environmentfor optimum activity and stability of the DNA polymerase

Dialent cations magnesium or manganese ions< generallyMg!" ) All thermostable DNA polymerases require free divalent

cations for activity) =ecause dN'P binds 1g/; the molarconcentration of 1g/;must e.ceed the molar concentration ofdN'Ps) >sually ")5 3/)5 m1 is used)

1onovalent cation potassium ions# 6tandard PCR buffercontains 50 m1 ?Cl


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PCR Cycle


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Three$ste! Cyclin% Process

+nitial denaturation

Denaturation

Annealing @.tension

inal e.tension

8old


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&enaturation (T#):

'he first step: thermal denaturation of the DNA sampleat #(#BoC for $0 s(" min

Double(stranded DNA template denature at a

temperature that is determined in part by their 7 ; Ccontent

'he higher the proportion of 7 ; C the higher the

temperature required to separate DNA strands

'he longer the DNA molecules the greater the timerequired at the chosen denaturation temperature to

separate the two strands completely


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+f the temperature for denaturation is too low or if

the time is too short only A'(rich regions of thetemplate DNA will be denatured

hen the temperature is reduced later in the PCR

cycles the template DNA will re(anneal into a fullynative condition

At #(#5oC Taqpolymerase can only endure for2$0(0 cycles without sustaining e.cessive damage


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'nnealin% (Ta) o" !rimers to tem!late &N'

+f the annealing temperature ,'a- is too high theoligonucleotide primers anneal poorly to the templateand the yield of amplified DNA is very low

+f 'a is too low non(specific annealing of primers mayoccur

Annealing is usually carried out $(5oC lower than thecalculated melting temperature ,'m-

'he length and nucleotides contents ,especially 7 ; C- of

the primers used in PCR determine the 'a


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'he time of annealing process ,2$0(B0s- also determineby the length and nucleotides contents of the primers


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tension o" Oli%onucleoti#e Primers

+s carried out at /(&oC

+n the first two cycles e.tension from one primerproceeds beyond the sequence complementary to the

binding site of the other primer

rom the third cycle onward this segment of DNA isamplified geometrically or e.ponentially

'he polymerisation rate of Taqpolymerase is 2/000nucleotidesEminutes at the optimal temperature ,/(&oC- but in e.periment e.tension is carried out for $

minute for eery $%%% &p of product


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*inal tension an# +ol#


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*inal tension an# +ol#

>sually after the last cycle of PCR an e.tensiontime of 5("0 min is carried out to allow thecompletion of all amplified products

Fastly the temperature is brought down to ("5oC until the sample is ta%en out


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Number o" Cycles

'he number of cycles required for amplificationdepends on the number of copies of template DNApresent at the beginning of the reaction

'he reaction proceeds until one of the componentsbecomes limiting

At least /5 cycles are required to achieve acceptablelevels of amplification of single(copy target sequences

>sually is between /5(0 cycles


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PCR Cycle

+nitial denaturation #5oC / min

Denaturation #5oC $0 s

Annealing 50(B0oC $0s

@.tension /oC " minE%b

inal e.tension /oC 5("0 min

8old ("5oC G

!'()%

cycles


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The Thermal Cycler


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'he thermal cycler heats and cools the reaction tubes toachieve the temperatures required at each step of the

reaction

1any modern thermal cyclers ma%e use of the Peltiereffect which permits both heating and cooling of the bloc%

holding the PCR tubes simply by reversing the electriccurrent

1ost thermal cyclers have heated lids to preventcondensation at the top of the reaction tube

Hlder thermo cyclers lac%ing a heated lid require a layer ofoil on top of the reaction mi.ture or a ball of wa. inside thetube


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,ummary

") Principles

/) Procedure

$) Components of PCR

) PCR Cycle


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End of Lecture 9Thank You

Lecture 9 PCR

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