Question

In addition to the amount of DNA, what other variables may need to be optimized in order for the proper amplification of a DNA region using PCR? Explain at least 2

Answer 1

In order for the proper amplification of a DNA region using PCR one must optimize

1. Designing of Primer- it includes Primer length , Optimal G-C content, Optimization of melting temperatures of primer. Single or dinucleotide repeats should be avoided. One can make use of primer designing softwares or online facilities available. It is adviced to run a BLAST to check the accuracy and specificity of primer. It will help us to avoid the amplification of speudogenes

2. Calculation of Metling point- It is inevitable to understand the melting point of the primer which is going to use for PCR amplification. A rough calculation can be done by using the formula Tm ≈ 4(G-C) + 2(A-T).. However, softwares to calculate the exact melting point based on the primer sequences are available

Other conditions to be optimized before doing PCR are

• Standerdization of PCR reagents such as dNTPs, DNA polymerase, Mg2+ etc
• Preparation of reaction mixture
• Setting up of denaturing and reannealing temperature
• Concentration of polymerases
• Extension temperature and extension time
• Use of purified and high quality DNA templates