PCR Technique

                                               PCR

What Is PCR -    PCR is Polymerase Chain Reaction used for making million copy of particular section of DNA . In 1983 Kary Mullis developed this technique used in variety of application like in medical case , criminal case , in diagnosis of genetic diseases and so on . For this work Mullis awarded  by Noble prize in 1993 . PCR is rapid , inexpensive and simple way of copying of DNA fragments by using smaller quantity of source of DNA material

 PCR Tools  - PCR can not be accomplish without this key tool -

•        Template DNA
•         Nucleotides ( dATP , dCTP , dGTP , dTTP )
•         Primer
•         Taq Polymerase Enzyme
•          Buffer  for stability of DNA polymerase .        

Working Of PCR - PCR is a chain reaction as it synthesize  DNA strands and act as template for further cycles . There are following stages to complete this process -

Denaturation -  In this stage the double stranded DNA is make single stranded by heating it at about 93 - 95 degree Celsius . This high temperature break the hydrogen bond between the two strands and due to this double stranded DNA becomes single stranded . Usually 15 minutes are required to complete this process .

Annealing -  In this step the temperature is reduced to 50 to 75 so that DNA primer is easily attach with the DNA template . A high temperature prevent primer and template to anneal . Primer are 20 -30 base long and are single stranded DNA . This act as starting point for DNA synthesis .

Extending - The temperature is raised to 72 so that Taq polymerase enzyme bind with the annealed primer .This enzyme is heat loving so that  it is very stable in high temperature. 72 degree temperature is enough for this enzyme to build complementary DNA strands . Taq polymerase work it ways along  the DNA   and adding complementary nucleotide   using the dNTP and other

component in the reaction mix . Very short duration is required for completing of this process .

These three stages are repeated multiple time to complete 20 - 40 cycles which doubling the DNA copies each time . Less than a hour is required to complete this reaction .
After the completion of this procedure the  quantity of DNA and size is checked by using Eletrophoresis method .

Application -

• PCR is very valuable for finding infectious disease like HIV , Hepatitis etc .
• PCR is also used by forensic departments for solving criminal disputes .
• PCR technique is  used for determine gene expressions .
• Mapping can be done with the help of PCR technique .
• PCR is helpful in analysis of  mutation that occur in many genetic diseases like cystic fibrosis , sickle cell anemia .  
•  The paternity issue is easily solved with this technique .

Limitation -  Sometime false results are come in the case of infectious disease so please follow conformation test .