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POLYMERASE CHAIN REACTION POLYMERASE CHAIN REACTION “ Hands on training  in Biotechnology” (2011) Centre of Excellence in Agri-Biotechnology, SVPUAT,Meerut,UP. R.Sujatha, Scientist B, New Delhi.
CONTENTS: ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
  History  OF PCR As is the photo copier a basic requirement in an office,so is the PCR machine in a molecular biology Laboratory !!!!!!!!! PCR is DNA replication in a test tube……..
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
DNA ?
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Requirements of PCR
STEPS INVOLVED: ,[object Object],[object Object],[object Object]
ANNEALING: ,[object Object],[object Object],[object Object],[object Object],[object Object]
EXTENSION: ,[object Object],[object Object],[object Object],[object Object]
NEW  AUTOMATED PCR  OLD PCR
Factors for Optimal PCR : ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Variations of PCR: ,[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object]
[object Object],- It is employed for amplification of RNA molecules .  - RT-PCR is widely used in expression profiling , to determine the expression of a gene or to identify the sequence of an RNA transcript.
[object Object],-Used to obtain 3' and 5' end sequence of cDNA transcripts.
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object]
[object Object],- It is used for synthesis of Single stranded DNA molecules useful for DNA sequencing -The two primers are used in the 100:1 ratio so that after 20-25 cycles of amplification one primer is exhausted thus single stranded DNA is produced in the next 5-10 cycles. ,[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
   Parameter PCR Gene cloning 1. Final result Selective amplification of specific sequence  Selective amplification of specific sequence  2. Manipulation In vitro In vitro and in vivo  3. Selectivity of the specific segment from complex DNA First step Last step 4. Quantity of starting material  Nanogram (ng)  Microgram (m) 5. Biological reagents required DNA polymerase  (Taq polymerase) Restriction enzymes, Ligase, vector. bacteria 6. Automation Yes No 7. Labour intensive No Yes 8. Error probability Less More 9. Applications More Less 10. Cost  Less More 11. User’s skill Not required Required 12. Time for a typical experiment Four hours Two to four days
Advantages: ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Limitations ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
PCR and  its types

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PCR and its types

  • 1. POLYMERASE CHAIN REACTION POLYMERASE CHAIN REACTION “ Hands on training in Biotechnology” (2011) Centre of Excellence in Agri-Biotechnology, SVPUAT,Meerut,UP. R.Sujatha, Scientist B, New Delhi.
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  • 3. History OF PCR As is the photo copier a basic requirement in an office,so is the PCR machine in a molecular biology Laboratory !!!!!!!!! PCR is DNA replication in a test tube……..
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  • 17. NEW AUTOMATED PCR OLD PCR
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  • 33.   Parameter PCR Gene cloning 1. Final result Selective amplification of specific sequence Selective amplification of specific sequence 2. Manipulation In vitro In vitro and in vivo 3. Selectivity of the specific segment from complex DNA First step Last step 4. Quantity of starting material Nanogram (ng) Microgram (m) 5. Biological reagents required DNA polymerase (Taq polymerase) Restriction enzymes, Ligase, vector. bacteria 6. Automation Yes No 7. Labour intensive No Yes 8. Error probability Less More 9. Applications More Less 10. Cost Less More 11. User’s skill Not required Required 12. Time for a typical experiment Four hours Two to four days
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