PCR is the technique forgenerating large quantities ofspecific DNA .PCR is a cell free amplificationtechnique to synthesizing multipleidentical copies of the DNA .It utilizes the principle of DNAreplication.
In 1993 Kary Mullis was awarded theNobel Prize for the discovery of PCR. In 1983 Karry mullisconceived the idea of PCR.Developed in 1984.
The double stranded DNA of interest is denaturedto separate into two individual strands.Each strand is then allowed to hybridize with aprimer (renature).The polymerase enzyme that starts synthesizing newstrandsThese three steps are repeated to get more copies ofDNA
copies of DNAsynthesisRenaturation orannealingdenaturation
Target DNA (100-35,000 bp in length)Primers (synthetic oligonucleotides of 17-30 nucleotides in length )Four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP)Thermo stable DNA polymerase that can withstandat a temperature upto 95 C (derived from Thermusaquaticus)
•Buffer solution, providing a suitable chemical environmentfor optimumactivity and stability of the DNA polymerase.•Divalent cations, magnesium or manganese ions; generallyMg2+ is used, but Mn2+ can be utilized for PCR-mediatedDNA mutagenesis, as higher Mn2+ concentration increasesthe error rate during DNA synthesis.• Monovalent cation potassium ions.
Major processes in PCRDenaturation:The temperature is raised at 94–98 C for 1minute to separatethe double stranded DNA.Renaturation:decrease the temperature at 55 C . This helps the primer tobind with target DNA . This step is also known as annealing.Synthesis :The initiation of DNA synthesis occurs at 3’- hydroxyl end ofeach primer. The primers are extended by joining the basescomplementary to DNA at 75 C.Note : This whole steps are considered as one cycle
Initializationstep: This step consists of heating the reaction to atemperature of 94–96 C (or 98 C if extremely thermostable polymerases areused), which is held for 1–9 minutes. It is only required for DNA polymerasesthat require heat activation by hot-start PCR.Denaturation step: This step is the first regular cycling event andconsists of heating the reaction to 94–98 C for 20–30 seconds. It causes DNAmelting of the DNA template by disrupting the hydrogen bonds betweencomplementary bases, yielding single-stranded DNA molecules.Annealing step: The reaction temperature is lowered to 50–65 C for 20–40 seconds allowing annealing of the primers to the single-stranded DNAtemplate. Typically the annealing temperature is about 3-5 degrees Celsiusbelow the Tm of the primers used. Stable DNA-DNA hydrogen bonds are onlyformed when the primer sequence very closely matches the templatesequence. The polymerase binds to the primer-template hybrid and beginsDNA synthesis.
Extension/elongation step: The temperature at this step depends on theDNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 C,and commonly a temperature of 72 C is used with this enzyme. At this stepthe DNA polymerase synthesizes a new DNA strand complementary to the DNAtemplate strand by adding dNTPs that are complementary to the template in 5 to 3direction, condensing the 5-phosphate group of the dNTPs with the 3-hydroxylgroup at the end of the nascent (extending) DNA strand. The extension timedepends both on the DNA polymerase used and on the length of the DNA fragmentto be amplified. As a rule-of-thumb, at its optimum temperature, the DNApolymerase will polymerize a thousand bases per minute. Under optimumconditions, i.e., if there are no limitations due to limiting substrates or reagents, ateach 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 temperatureof 70–74 C for 5–15 minutes after the last PCR cycle to ensure that any remainingsingle-stranded DNA is fully extended.Final hold: This step at 4–15 C for an indefinite time may be employed forshort-term storage of the reaction.
Thermo cyclerThis whole process was done by using an automated machine called asthermo cycler. It can raises and lowers the temperature automatically.The PCR is commonly carried out in a reaction volume of 10–200 μl insmall reaction tubes (0.2–0.5 ml volumes) in a thermal cycler.The thermal cycler heats and cools the reaction tubes to achieve thetemperatures required at each step of the reactionModern thermo cyclerAn older model three-temperaturethermal cycler for PCR
01020304050607080901001 2 3 4 5 6 7temperaturetimetemperature vs time
Nested PCRNested primers increases thespecificity and selectively amplifiesthe target DNA.Inverse PCRCan be study the unknownsequences using known sequence.Anchored PCRthis is particularly useful when thesequence surrounding the target isnot known. It can be done by using
Reverse transcription PCRThe mRNA converted to cDNA by reversetranscriptase , this cDNA serve as thetemplate for PCRReal time PCRCommonly used technique for measuringthe quantity ofDNA by employing fluorescencecompound ethidium bromide.Asymmetric PCRThis technique can be used for thesynthesis of single stranded DNA, particularly used for DNA sequencing.
PCR in clinical diagnosis:Prenatal diagnosis of inheriteddiseases:Using chorionic villus samples orcell from amniocentesis. Thusdiseases like sickle-cellanemia, β- thalassemia can bedetected by PCR.Diagnosis of retroviral andbacterial infections:PCR from cDNA is valuable tool for
PCR in comparative study ofgenomes:The differences in the genomescan be measured afterelectrophoresis. The closelyrelated organisms can givesimilar bands.PCR is very useful in the study ofevolutionary biology, morespecifically referred asphylogenetics.
PCR IN FORENSICS:A single molecule of DNA from anysources like blood ,hair, small tissueetc can be amplified by PCR.The PCR is useful in the DNA fingerprinting technology.