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Presented by-AnshikaBSc Hons

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What is PCR?• PCR targets and amplifies

a specific region of a DNA strand.

• It is an in-vitro technique to generate large quantities of a specified DNA.

• PCR is ‘photocopier.’

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HISTORY

• Kary B. Mullis, developed PCR in 1985 and was awarded the Nobel Prize for Chemistry in 1993.

• PCR machine =thermocycler

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Thermocycler

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REQUIREMENTS

• DNA Template• Primers• Taq polymerase• Deoxynucleoside

triphosphates(dNTPs)• Buffer solution• Divalent

cations(eg.Mg2+)

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STEP INVOLVED:

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ANNEALING:

• The annealing step allows the hybridisation of the two oligonucleotide primers, which are present in excess, to bind to their complementary sites that flank the target DNA.

• The annealed oligonucleotides act as primers for DNA synthesis, since they provide a free 3’ hydroxyl group for DNA polymerase.

• TEMPERATURE :45-60°C

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Taq DNA polymerase• The availability of a thermostable DNA

polymerase enzyme isolated from the thermophilic bacterium Thermus aquaticus found in hot springs provided the means to automate the reaction.

Taq DNA polymerase has a temperature optimum of 72°C and survives prolonged exposure to temperatures as high as 96°C and so is still active after each of the denaturation steps.

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Types of PCR

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Inverse PCRAmplification of DNA

of unknown sequence carried out from known sequence.

identification of sequences flanking transposable elements

identification of genomic inserts

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REVERSE TRANSCRIPTION PCR (RT-PCR)

• for amplifying DNA from RNA.

• Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR.

• Some thermostable DNA polymerases used in the PCR such as Tth have a reverse transcriptase activity under certain buffer conditions.

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Quantitative real time PCR(Q-RT PCR)• It quantitatively measures

starting amounts of DNA, cDNA or RNA.

• Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample.

• QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGen to measure the amount of amplified product in real time.

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22THANK YOU