PCR-SlideShare

This ppt is a brief introduction of PCR i.e. POLYMERASE CHAIN REACTION.Topic covered-basic introduction,steps involved in the reaction,types of PCR.

1. Presented by- Anshika BSc Hons 1

2. 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.’ 2

3. HISTORY Kary B. Mullis , developed PCR in 1985 and was awarded the Nobel Prize for Chemistry in 1993 . PCR machine = thermocycler 3

4. T hermocycler 4

5. R EQUIREMENTS DNA Template Primers Taq polymerase Deoxynucleoside triphosphates( dNTPs ) Buffer solution Divalent cations (eg.Mg2+) 5

6. STEP INVOLVED: 6

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9. 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 9

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12. 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. 12

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16. Types of PCR 16

17. Inverse PCR Amplification of DNA of unknown sequence carried out from known sequence. identification of sequences flanking transposable elements identification of genomic inserts  17

18.   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 . 18

19. 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. 19

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22. THANK YOU 22