Polymerase Chain Reaction (PCR) Technology for M.TB

Polymerase Chain Reaction (PCR) is a laboratory technique to amplify Deoxyribose Nucleic Acid (DNA).

The PCR mix consists of:

• MgCl₂ :1.5 - 6 mM
• Buffer (pH 8.3 - 8.8)
• DNA polymerase (Taq polymerase):  0.5 - 2.5 U
• Target DNA: <1µg  
• Primers: Short DNA sequences to select the region to be amplified

During PCR, the temperature of the PCR mix is repeatedly raised and lowered to help the DNA polymerase enzyme replicate the target DNA sequence in the presence of the primer (Figure 1) and produce multiple copies in a few hours (Figure 2).

Steps in PCR:

• Denaturation of the DNA into single strands (94-95ºC)
• Annealing of primers to each strand for new strand synthesis (58-65ºC)
• Extension of the new DNA strands (72ºC)

The test is performed in a thermal cycler machine that maintains a different temperature required during the different PCR steps.

Figure 1: First Cycle in Thermal Cycler Machine

Figure 2: Exponential Amplification of Target Gene

Resources

• GLI Training Package on LPA, 2012.​

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