The PCR primers D1S80(F) and D1S80(R) amplify DNA from a specific region of huma

Question

The PCR primers D1S80(F) and D1S80(R) amplify DNA from a specific region of human chromosome 1. The target region contains a variable number of tandem repeats (VNTR) that is highly informative in DNA fingerprinting studies. The expected PCR product can vary from 340 - 650 base pairs in length. The sequence of the primers is given below: D1S80 (F) 5' - GAA ACT GGC CTC CAA ACA CTG CCC GCC GTC CA D1S80 (R) 5'-GTC TTG TTG GAG ATG CAC GTG CCC CTT GCA GG Given the size of the expected PCR product and the sequence of the primers, design a thermocycle program to amplify this region of DNA. Be sure to list the stages required in the proper order they need to be executed. For each stage indicate the number of times it must be executed, and provided detailed information on the time temperature required.

Explanation / Answer

Answer-

Forward Primer for PCR is length= 32 base pairs, G= 6, C=14, A=8, T=4

Reverse primer is length = 32 base pair, G=11, C=8, A=4, T=9

Tm value = 4 (G+C) + 2 (A+T) if the primer base pair is less than 16. but here the primer length is 32. Hence use the formula Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)

And to get the amplicon size of 350 to 650 base pair approxmately 25 to 30 cycle is required.

Take the human genomic DNA of 1 ng - 1 ug

PCR conditions are

1 cycle - 95 deg Centtigrade for 2 minutes (denaturation initial)

30 cycles [ 95 deg C for 15 seconds

65 deg C for 15 secpnds

68 deg C for 45 seconds

1 cycle at 68 deg C for 5 minutes ( for complete replication of all templets)

1 cycle at 4 deg C or infinite time ( for storage , until PCR product get used)

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