Some of your classmates just realized that when setting up the initial PCR react

Question

Some of your classmates just realized that when setting up the initial PCR reaction, the master mix MMNP (containing the nested set of primers was used). Then, for the second “nested” PCR, the group used the master mix MMIP (containing the initial set of primers). Do you expect this series of reactions to have yielded the same PCR product(s) as if they had performed the reaction correctly? As you explain your answer, consider,

a. The location of the primer sets relative to each other

b. Whether the thermal cycler programs are the same or different

Explanation / Answer

Ans) For the PCR reaction which is polymerase chain reaction, we need to add a master mix MMNP which is nothing but a mixture of the Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of DNA templates. In PCR the main 3 steps involved are the (1) denaturation of the template into single strands; (2) annealing of primers to each original strand for new strand synthesis; and (3) extension of the new DNA strands from the primers. When we use the primers in the case of the primer is a short strand of RNA or DNA (generally about 18-22 bases) that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process.

(a)Now the location of the primer is done on the nucleotide of the DNA which happens during the elongation phase of the system. In that system,DNA polymerase binds to the 3´ ends of each primer and, using the single-stranded DNA as a template, incorporates new nucleotides from a 5´ to 3´ direction. The length of this stage depends upon the length of the target sequence. The primer location is very intensive and specific to the targetted sequence of the DNA strands. Along with this the degenerate PCR primers can be created such that the primers contain a mixture of bases at specific positions.

(b) The thermal cycle process of the whole process is same and a wide range of PCR cycling protocols have been used for various molecular biology applications. These cycles are basically important to give the precise result at the DNA level of the strands

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