What is Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is used to multiply and make millions of copies of DNA (also called amplification of DNA). It is a technique used to amplify small and targeted segments of DNA to produce millions of copies of a specific gene fragment. It is an efficient and cost effective molecular technique.

Raw material used for PCR

For in vitro synthesis of DNA we need following raw material to make DNA.

Primers Definition

(Forward and Reverse) These are short,artificial pieces of single stranded DNA usually around 2- 30 nucleotides in length. They are used to identify specific region to be copied. They attach to the beginning and the end region to be amplified.

Taq Polymerase 

These are enzymes which attach nucleotides with DNA thereby, copying the DNA. These enzymes are special as they can work in high temperature which is necessary for PCR procedure. Actually they are separated from bacteria Thermus aqaticus. You can buy taq polymerase here.

DNA template

It is a sample DNA which contain the target sequence to amplify.

PCR Buffer

which provide suitable chemical environment for DNA Polymerase. It also act as a co-factor for the enzyme. e.g 10mM Tris at pH 8.3, 50mM KCl,1.5-2.5 mM MgCl, DMSO, Glycerol formamide etc.

Deoxynucleoside triphosphates (dNTPs)

dNTPs consist of dATPs, dCTPs, dGTPs, dTTPs. They are building blocks of new DNA strand.

Thermocycler

PCR is carried out in a machine called Thermocycler, which can heat and cool the reaction tubes to achieve require temperature.

Steps of PCR

Polymerase Chain Reaction involves following steps

  1. Denaturing (at 94-96°C)
  2. Annealing (~68°C)
  3. Elongation (at 72°C)

Lets explain these steps one by one;

Denaturing: (DNA Separation) It is a separation of DNA strand. When we heat it to 94-96°C, the Hydrogen bonds between the strands break down result in releasing two strands.

Annealing: (Primer binding) In this step primer anneal or attach to the both DNA templates at 50-65°C. Primer recognize specific target DNA which we need to be copied.

Elongation: (Attachment of Nucleotides) At 72°C the Taq Polymerase attach itself to the primer and help it to collect and join new nucleotides to make a new strand of DNA.

Once the process completes, 1 DNA is converted in two copies in 1-3 min. This three step process repeat and repeat making millions of copies of DNA in 45 min.

Target copy hours

 

Polymerase Chain Reaction Cycles vs Target Copies

The copies of the DNA doubled in each cycle so after “n” cycles you have 2^n (2 to the nth power). For example after 10 cycles you have 1024 copies and after 20 cycles you have 1 million copies etc.

Analysis of PCR Products

There are many techniques used to analyze PCR products.

  1. Agarose Gel Electrophoresis
  2. Labelled Probe

Gel Electrophoresis

As name suggests Gel Electrophoresis contains a gel (made up of a polysaccharide called Agarose) The gel is placed in a tub containing a buffer (water with some salt in it). In this technique we used to separate mixture of DNA fragments or other macromolecules (e.g RNA, Proteins) according to their molecular size and charge present on them. At One end there are pocket like wells where we put DNA fragments. One end of a box is hooked to a positive end of a battery and other end is attached with a negative end as shown in the following picture.

Gel

This is how DNA fragments separated by the size. The shorter pieces travel faster than the larger ones.

Once the DNA fragments are separated. we can see and analyze the results by finding which size of DNA we’ve found. To make DNA particles visible we need to stain with a DNA binding dye and place under UV transilluminatikon. If targeted band is present it indicates otherwise don’t. You can find DNA Classroom kit here.

Labelled Probe

These are DNA/RNA fragments of variable length (100-1000 nucleotides long) is used to detect PCR based genes product (also called amplicon). By nature a probe can eleminate radioactive, fluorometric, colorometric or chemilluminiscent signals. It helps to visualize a product. Moreover, it provide specificity by highlighting specific targeted sequence (amplicons). DNA Labelled probe is available here.

Types of PCR

There are many types of PCR. Which include;

  1. Real time PCR
  2. Conventional PCR
  3. Nested-seminested PCR
  4. Multiplex PCR
  5. Quantitative PCR
  6. Touch down PCR
  7. Arbitrary PCR
  8. Inverse PCR
  9. Allele specific PCR
  10. Asymmetric PCR
  11. Core sample PCR
  12. Dial-out PCR
  13. Digital PCR
  14. Assembly PCR
  15. Traditional PCR
  16. Hot Start PCR
  17. In-silico PCR
  18. Inter sequence PCR
  19. Ligation-mediated PCR
  20. Methylation-specific PCR
  21. Miniprimer PCR
  22. Nano particle PCR
  23. Overlap extension PCR
  24. Quantitative PCR
  25. Solid phase PCR
  26. Suicide PCR
  27. Thermal asymmetric interplaced PCR
  28. Semiquantitative PCR
  29. Colony PCR
  30. After exponentional PCR
  31. Qualitative PCR

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