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In: Biology

In addition to the amount of DNA, what other variables may need to be optimized in...

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

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Expert Solution

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

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