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PCR
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AI-enhanced description

The polymerase chain reaction (PCR) is a

technique used to amplify a specific DNA
sequence. It involves cycling between heating
and cooling steps to denature and replicate
DNA. The reaction requires DNA template,
primers, DNA polymerase, nucleotides, and
bu!er. During each cycle, the DNA denatures,
primers anneal, and the polymerase extends the
DNA. This exponential amplification allows
millions of copies of the target sequence to be
generated from a small initial sample. PCR has
many applications in medicine, research, and
forensics.
Read less

Technology

Microbiology

1.InitializationStep:

Itisthefirststepofthecyclewhichconsistsofraisingthetemperatureofthereactionto94-96
°Cor98°Cifextremelythermostablepolymerasesareused,whichisheldfor1-9minutes.This
processactivatestheDNApolymeraseusedinthereaction.

2.Denaturation

Thisstepinvolvesheatingthereactionmixtureto94°Cfor15-30seconds.Duringthis,the
doublestrandedDNAisdenaturedtosinglestrandsduetobreakageinweakhydrogenbonds.

3.Annealing:

Thereactiontemperatureisrapidlyloweredto54-60°Cfor20-40seconds.Thisallowsthe
primerstobind(anneal)totheircomplementarysequenceinthetemplateDNA.
4.Elongation:

Alsoknownatextension,thisstepusuallyoccursat72-80°C(mostcommonly72°C).Inthis
step,thepolymeraseenzymesequentiallyaddsbasestothe3'eachprimer,extendingtheDNA
sequenceinthe5'to3'direction.Underoptimalconditions,DNApolymerasewilladdabout
1,000bp/minute.

Withonecycle,asinglesegmentofdouble-strandedDNAtemplateisamplifiedintotwo
separatepiecesofdouble-strandedDNA.Thesetwopiecesarethenavailableforamplificationin
thenextcycle.Asthecyclesarerepeated,moreandmorecopiesaregeneratedandthenumber
ofcopiesofthetemplateisincreasedexponentially.

5.Finalelongation.
Thisstepisperformedatatemperatureof70-74degreecentigradefor5-15minutesafterthelast
PCcycletoensurethatanyremainingsingle-strandedDNAisfullyextended.

6.Finalhold.

Inthisstepthemixtureisallowedtocooltoatemperatureof4-15degreecentigradeforshort-
termstorageofthereaction.

AmjadKhanAfridi

material,hair,bones,mummifiedtissues.
3.Instudyofgeneexpressionanalysis,PCbasedmutagenesis
4.InHumangenomeprojectforaimtocompletemappingandunderstandingofallgenesof
humanbeings.

Components
Thebasiccomponentsandreagentsrequiredtosetupa100ulPCreactionare:

1.Microfugetube.

Thesearesmallcylindricalplasticconicalcontainerswithconicalbottomswithasnapcap.They
aremadeupofpolypropylene,thuscanwithstandawiderangeoftemperature.

2.Thermalcycler.
3.ItisanapparatususedtoamplifysegmentsofDNA.Ithasathermalblockwithholes
wheretubesholdingthePCreactionmixturescanbeinserted.Thecyclerworksonthe
principleofPeltiereffect,whichraisesandlowersthetemperatureoftheblockinapre-
programmedmannerbyreversingtheelectriccurrent.Thin-walledreactiontubespermit
favorablethermalconductivitytoallowforrapidthermalequilibration.
4.DNAtemplate.

Thereactionsolutionshouldcontainatleast(le5-letargetmolecules).

5.Primer.

Theseareoligonuclotidesthatdefinethesequencetobeamplified.Twoprimerthatare
complementarytothe3'(threeprimeendsofeachofthesenseandanti-sensestrandofthe
DNAtarget(Tm52-58degreecentigradepreferred).Primerswithmeltingtemperaturesabove
65degreecentigradehaveatendencyforsecondaryannealing.TheGCcontent(thenumberof
G'sandC'sintheprimerasapercentageofthetotalbases)ofprimershouldbe40-60%.
FormulaforprimerTmcalculation:

MeltingTemperatureTm(oK)={AH/AS+RIn(C)),OrMeltingTemperatureTm(oC)={AH/
AS+RIn(C)}-273.15where

AH(kcal/mole):HistheEnthalpy.Enthalpyistheamountofheatenergypossessedby
substances.AHisthechangeinEnthalpy.IntheaboveformulatheAHisobtainedbyaddingup
allthedi-nucleotidepairenthalpyvaluesofeachnearestneighborbasepair.

AS(kcal/mole):Sistheamountofdisorderasystemexhibitsiscalledentropy.ASischangein
Entropy.Hereitisobtainedbyaddingupallthedi-nucleotidepair'sentropyvaluesofeach
nearestneighborbasepair.AnadditionalsaltcorrectionisaddedastheNearestNeighbor

AmjadKhanAfridi

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Explained (2 in 1)

PCR
1. 1 Amjad Khan Afridi PolymeraseChainReaction(PCR):
Principle, Procedure, Components, Types , Applications and PCR
Stages The polymerase chain reaction (PCR) is a laboratory
technique for DNA replication that allows a “target” DNA
sequence to be selectively amplified. PCR can use the smallest
sample of the DNA to be cloned and amplify it to millions of
copies in just a few hours. Discovered in 1985 by Kerry Mullis, PCR
has become both and essential and routine tool in most
biological laboratories. Principle of PCR The PCR involves the
primer mediated enzymatic amplification of DNA. PCR is based
on using the ability of DNA polymerase to synthesize new strand
of DNA complementary to the o!ered template strand. Primer is
needed because DNA polymerase can add a nucleotide only onto
a preexisting 3′-OH group to add the first nucleotide. DNA
polymerase then elongate its 3 end by adding more nucleotides
to generate an extended region of double stranded DNA.
Components of PCR The PCR reaction requires the following
components: 1. DNA Template : The double stranded DNA
(dsDNA) of interest, separated from the sample. 2. DNA
Polymerase : Usually a thermostable Taq polymerase that does
not rapidly denature at high temperatures (98°), and can function
at a temperature optimum of about 70°C. 3. Oligonucleotide
primers : Short pieces of single stranded DNA (o#en 20-30 base
pairs) which are complementary to the 3’ ends of the sense and
anti-sense strands of the target sequence. 4. Deoxynucleotide
triphosphates : Single units of the bases A, T, G, and C (dATP,
dTTP, dGTP, dCTP) provide the energy for polymerization and the
building blocks for DNA synthesis. 5. Bu!er system : Includes
magnesium and potassium to provide the optimal conditions for
DNA denaturation and renaturation; also important for
polymerase activity, stability and fidelity. Procedure of PCR All the
PCR components are mixed together and are taken through series
of 3 major cyclicreactions conducted in an automated, self-
contained thermocycler machine.
2. 2 Amjad Khan Afridi 1. Initialization Step: It is the first step of
the cycle which consists of raising the temperature of the reaction
to 94–96 °C or 98 °C if extremely thermostable polymerases are
used, which is held for 1–9 minutes. This process activates the
DNA polymerase used in the reaction. 2. Denaturation : This step
involves heating the reaction mixture to 94°C for 15-30 seconds.
During this, the double stranded DNA is denatured to single
strands due to breakage in weak hydrogen bonds. 3. Annealing :
The reaction temperature is rapidly lowered to 54-60°C for 20-40
seconds. This allows the primers to bind (anneal) to their
complementary sequence in the template DNA. 4. Elongation :
Also known at extension, this step usually occurs at 72-80°C (most
commonly 72°C). In this step, the polymerase enzyme
sequentially adds bases to the 3′ each primer, extending the DNA
sequence in the 5′ to 3′ direction. Under optimal conditions, DNA
polymerase will add about 1,000 bp/minute. With one cycle, a
single segment of double-stranded DNA template is amplified
into two separate pieces of double-stranded DNA. These two
pieces are then available for amplification in the next cycle. As
the cycles are repeated, more and more copies are generated and
the number of copies of the template is increased exponentially.
5. Final elongation. This step is performed at a temperature of 70-
74 degree centigrade for 5-15 minutes a#er the last PCR cycle to
ensure that any remaining single-stranded DNA is fully extended.
6. Final hold. In this step the mixture is allowed to cool to a
temperature of 4-15 degree centigrade for short- term storage of
the reaction.
3. 3 Amjad Khan Afridi Types of PCR In addition to the
amplification of a target DNA sequence by the typical PCR
procedures already described, several specialised types of PCR
have been developed for specificapplications. 1. Real-time PCR 2.
Quantitative real time PCR (Q-RT PCR) 3. Reverse Transcriptase
PCR (RT-PCR) 4. Multiplex PCR 5. Nested PCR 6. Long-range PCR 7.
Single-cell PCR 8. Fast-cycling PCR 9. Methylation-specific PCR
(MSP) 10. Hot start PCR
4. 4 Amjad Khan Afridi 11. High-fidelity PCR 12. In situ PCR 13.
Variable Number of Tandem Repeats (VNTR) PCR 14. Asymmetric
PCR 15. Repetitive sequence-based PCR 16. Overlap extension
PCR 17. Assemble PCR 18. Intersequence-specific PCR(ISSR) 19.
Ligation-mediated PCR 20. Methylation –specifin PCR 21.
Miniprimer PCR 22. Solid phase PCR 23. Touch down PCR, etc
Applications of PCR Some common applications of PCR in various
fields can be explained in following categories. Medical
Applications: 1. Genetic testing for presence of genetic disease
mutations. Eg: hemoglobinopathies, cysticfibrosis, other inborn
errors of metabolism. 2. Detection of disease causing genes in
suspected parents who act as carriers. Study of alteration to
oncogenes may help in customization of therapy 3. Can also be
used as part of a sensitive test for tissue typing, vital to organ
transplantation genotyping of embryo 4. Helps to monitor the
gene in gene therapy Infectious disease Applications: Analyzing
clinical specimens for the presence of infectious agents, including
HIV, hepatitis, malaria, tuberulosis etc. Detection of new
virulent subtypes of organism that is responsible for epidemics.
Forensic Applications: Can be used as a tool in genetic
fingerprinting. This technology can identify any one person from
millions of others in case of : crime scence, rule out suspects
during police investigation, paternity testing even in case of
avaibility of very small amount of specimens ( stains of blood,
semen, hair etc) Researchand Molecular Genetics: 1. In genomic
studies: PCR helps to compare the genomes of two organisms
and identify the di!erence between them. 2. In phylogenetic
analysis. Minute quantities of DNA from any source such a
fossilized
5. 5 Amjad Khan Afridi material, hair, bones, mummified tissues.
3. In study of gene expression analysis, PCR based mutagenesis 4.
In Human genome project for aim to complete mapping and
understanding of all genes of human beings. Components The
basic components and reagents required to set up a 100 ul PCR
reaction are: 1. Microfuge tube. These are small cylindrical plastic
conical containers with conical bottoms with a snap cap. They are
made up of polypropylene, thus can withstand a wide range of
temperature. 2. Thermal cycler. 3. It is an apparatus used to
amplify segments of DNA. It has a thermal block with holes where
tubes holding the PCR reaction mixtures can be inserted. The
cycler works on the principle of Peltier e!ect, which raises and
lowers the temperature of the block in a pre- programmed
manner by reversing the electric current. Thin-walled reaction
tubes permit favorable thermal conductivity to allow for rapid
thermal equilibration. 4. DNA template. The reaction solution
should contain at least (1e5-1e6 target molecules). 5. Primer.
These are oligonucleotides that define the sequence to be
amplified. Two primer that are complementary to the 3′ (three
prime) ends of each of the sense and anti-sense strand of the DNA
target (Tm 52-58 degree centigrade preferred). Primers with
melting temperatures above 65 degree centigrade have a
tendency for secondary annealing. The GC content (the number
of G’s and C’s in the primer as a percentage of the total bases) of
primer should be 40-60%. Formula for primer Tm calculation:
Melting Temperature Tm(oK) = {ΔH/ ΔS + R ln(C)}, Or Melting
Temperature Tm(oC) = {ΔH/ ΔS + R ln(C)}-273.15 where ΔH
(kcal/mole): H is the Enthalpy. Enthalpy is the amount of heat
energy possessed by substances. ΔH is the change in Enthalpy. In
the above formula the ΔH is obtained by adding up all the di-
nucleotide pair enthalpy values of each nearest neighbor base
pair. ΔS (kcal/mole): S is the amount of disorder a system exhibits
is called entropy. ΔS is change in Entropy. Here it is obtained by
adding up all the di-nucleotide pair’s entropy values of each
nearest neighbor base pair. An additional salt correction is added
as the Nearest Neighbor
6. 6 Amjad Khan Afridi parameters were obtained from DNA
melting studies conducted in 1M Na+ bu!er and this is the default
condition used for all calculations. ΔS (salt correction) = ΔS (1M
NaCl) + 0.368 x N x ln([Na+]) Where N is the number of nucleotide
pairs in the primer (primer length -1). [Na+] is salt equivalent in
mM. The primer annealing temperature is defined by the formula:
Ta = 0.3 x Tm (primer) + 0.7 Tm (product) -14.9 where, Tm(primer)
= Melting Temperature of the primers Tm(product) = Melting
temperature of the product 6. Tris-HCl. The recommended bu!er
solution is 10 to 50 mM Tris-HCl (pH 8.3-8.8) at 20 degree
centigrade. 7. MgCl2. It is the cofactor of the enzyme. It is
beneficial to optimize the magnesium ion concentration. The
magnesium ion a!ects the primer annealing, strand dissociation
temperatures of template and PCR product, product specificity,
formation of primer-dimer artifacts and enzymatic activity and
fidelity. Taq DNA polymerase requires free magnesium that binds
to template DNA, primers, and dNTPs. 8. KCl. KCl is to be used for
the reaction to facilitate primer annealing. 9. Gelatin or bovine
serum. Aautoclaved gelatin or nuclease-free bovine serum
albumins are included to help stabilize the enzyme. 10. Distilled
water. Autoclaved distilled water was used. The volume depends
on the reaction. 11. Deoxyneuclotide triphosphates. These are the
DNA building blocks. Dntp (TTP-thymidine triphosphate), dCTP
(deoxycyctidine triphosphate), dATP (deoxyadenosine
triphosphate) and dGTP (deoxyguanosine triphosphate) solutions
neutralized to pH 7.0. Primary stock solution are diluted to 10
mM, aliquoted, and
7. 7 Amjad Khan Afridi stored at -20 degree C. A working stock
containing 1 mM each dNTP is recommended. The stability of
dNTPs during repeated cycles of PCR is such that approximately
50% remains as dNTP a#er 50 cycles (Corey Levevenson, personal
communication). dNTP concentrations between 20 and 200 uM is
best for the reaction. The 4 dNTP should be at equivalent
concentrations to minimize mis-incorporation error. 12. DNA
polymerase. It is an enzyme that catalyzes the reaction. Taq DNA
polymerase isolated from Thermus aquaticus growing in hot
springs acts best at 72 degree centigrade and the denaturation
temperature of 90 degree centigrade does not destroy its
enzymatic activity. Other thermostable enzyme like Pflu DNA
polymerase isolated from Pyrococcus furiosus and Vent
polymerase isolated from Thermococcus litoralis, were
discovered and were found to be more e!icient. A recommended
concentration of Taq polymerase (Perkin-Elmer Cetus) is between
1 and 2.5 units (SA=20 units/pmol) per 100 uL reaction. However
enzymatic activity will vary with respect to individual target
templates or primers. To set up a 25 or 50 uL reaction the
concentration of the reagent are as follows: Reagent 10X bu!er
2.5 uL 5 uL dNTP 0.5 1 Forward primer 1 2 Reverse primer 1 2 Taq
polymerase 0.15 0.3 Water 18.85 uL 38.7 uL DNA (30-50 ng) 1 1
Total volume 25 uL 50 uL
8. 8 Amjad Khan Afridi PCR Stages 1. Exponential amplification.
As a result of each cycle, the number of copies of the desired
segment becomes twice the number present at the end of the
previous cycle. The more times the three PCR cycles are repeated
the more DNA you can obtain. This is because every cycle of a
PCR reaction theoretically doubles the amount of target copies,
so we expect a geometric amplification. In other words PCR is an
exponential process. One could use this formula to calculate the
theoretical output of any input: Y = X (1 + e!iciency) n Y=amount
of amplification target X=input copy number n =number of cycles
E!iciency factor is given for each cycle in the kit 2. Leveling o!
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. 3. Plateau stage. The term
“plateau e!ect” is used to describe the attenuation in the
exponential rate of product accumulation that occurs during late
PCR cycles. The plateau e!ect is a!ected by: Utilization of
substrates (dNTPs or primers). Stability of reactants (dNTPs or
enzyme). End-product inhibition (pyrophosphate, duplex DNA).
Competition for reactants by nonspecific products or primer-
dimer. Reannealing of specific product at concentrations above
1E8 M (may decrease the extension rate or processivity of Taq
DNA polymerase or cause branch-migration of products strands
and displacement of primers. Incomplete denaturation/strand
separation of product at high concentration.

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