In: Biology
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
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