PCR

Polymerase Chain Reaction: Basic idea and principle of testing

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Full name of PCR is Polymerase Chain Reaction (PCR). Using an enzyme Taq polymerase, DNA or required segments of DNA can be copied in a chain reaction. Within a span of 2-3 hours, millions of copies of DNA can be done.

The process was first developed by American biochemist Kary Mullis in 1983, and he was awarded Nobel Prize in 1993 for that.

PCR is used in molecular biology to make many copies of small section of DNA or a gene- thus ‘Amplify’ it.

Ref: genes by PCR. Cycle 4. Cycle 3. The gene of interest. Cycle 2. Cycle 1. Cycle 36. Reem Sallam, MD, PhD. 22 = = 8. 24= = 68billion.

It is a common tool used in medical and biological research lab for processing DNA for detecting the presence or absence of a GENE, also to identify pathogens during infection by identifying it’s DNA.

PCR also used in forensic purpose for identification.

INGREDIENTS REQUIRED

  • DNA template to be copied
  • Primers- the short stretches of DNA, that initiate the recation by binding with either side of separated DNA.
  • DNA nucleotide base- also known as dNTPs (A,C,G&T) the building blocks of DNA thet are needed to construct new strands of DNA.
  • Taq Polymerase enzyme.
  • Buffer to ensure right environment for processing.
  • Machine that control temperature by heating and cooling. The machine is calle THERMOCYCLER.

STEPS

  1. Denaturing : Selected double stranded DNA is separated into two single strand by applying heat.
  2. Temperature is raised to 90-95 degree centigrade,
  3. Annealing: Temperature is lowered. In low temperature DNA primers attach to the template DNA.
  4. Extending: Temperature is raised and enzyme Taq Polymerase is added, which form new strand of DNA.

The process is repeated 20-30 times, each time DNA is doubled, and millions of copied of DNA are thus formed.

PCR

In high speed machine, it may take less than a hour and usually takes 2-3 hoyrs.

After PCR has been completed, by means of ELECTROPHORESIS, quantity and size of DNA can be assayed.

What happens?

Denaturing

  • Temperature is raised to 90-95 degree centigrade. Hydrogen bonds between bases of two strands of template DNA break and two strand separate.
  • These strands act as template for the production of new strands of DNA. DAN must separate completely and till then temperature should be maintained at this stage. It usually takes 15-30 seconds.

Annealing

  • Temperature is lowered to 50-65 degree Celsius and at this temperature primer is attached to a specific location on the single stranded template DNAby hydrogen bonding.
  • Primer are single stranded RNA or DNA segment of 25-30 base length. This primers are complementary to short section of DNA of each end to be copied.
  • Primer serve as starting point of DNA synthesis and Taq Polymerase only add base to a double strand DNA increasing its length. Thus a new complementary strand of DNA form from loose DNA bases.
  • Without bonding of primer, polymerase has no function and polymerase enzyme can attach after binding of primer.
  • Two separated strand DNA are complementary and run in opposite direction, thus there are two primer, forward and backward. This steps usually takes 10-30 seconds.

Extending stages or extension

  • Heat again increased to 72 degree Celsius. Taq polymerase enzyme is added which add DNA bases and made new DNA.
  • Taq polymerase enzyme is collected from bacteria Thermusaquaticus, that normally lives in hot springs so can tolerate temperature above 80 degree Celsius. Other bacterial polymerase or human polymerase cannot stand that high temperature.
  • Taq polymerase attach to primer and then add DNA base to single starnd one by one.
  • The result is a brand new double strand of DNA and a double stranded DNA.
  • The process to copy 1000 DNA base (1Kb) takes around one minute.  Total time depends on length of DNA.
  • All the process are repeated 20-40 times. From a single copy, after 25 doubling, 16 million and after 30 doubling 563 million of DNA can be copied or ampilified.

Note

The PCR technology are still developing. New mwthods and refinement are being developed and used specially when quantification of DNA sample is needed.

New methods include

  • Real time PCR or quantitative PCR (qPCR)
  • Digital PCR (dPCR)- the most recent newer more refined process.

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