Question

The following is based on the DNA sequence and one of the oligos from the previous problem set, which are reproduced below:
5'...GGAGCTTCATGCTAGTTGCAATAGC..[1,150 bp]..CGTGGCACGTATAGCGCTATCATTA...3'
      oligo-B: CATGCTAGTTGC     
a) If oligo-B is used as a primer for (Sanger) DNA sequencing, which type of dideoxynucleotide would be found on the “first” (shortest) chain to terminate synthesis?
b) If oligo-B is used a sequencing primer, what the total length (number of nucleotides) of the SHORTEST DNA chain that would have a dideoxy-G (ddG, 2',3'-dideoxyguanosine) at its end?

Answer 1

Answer a: If oligo-B is used as a primer for (Sanger) DNA sequencing, the type of dideoxynucleoside that would be found on the “first” (shortest) chain to terminate synthesis is ddTTP (2′,3′-Dideoxythymidine 5′-Triphosphate). The first or the shortest chain would be of two nucleotides long having dGTP and ddTTP. For the replication of DNA, the deoxynucleosides (dNTPs) are required as the raw materials. Dideoxynucleosides (ddNTPs) are the chain elongating inhibitors of DNA polymerisation. In these nucleotides, the ribose sugar lacks the hydroxyl group in both 2’ and 3’ position. Incorporation of ddNTPs in the elongating DNA sequence during the process of normal replication (using dNTPs) cause chain termination. Therefore, the result is the production of DNA sequences of different lengths. This is the principle of Sanger DNA sequencing.

Answer b: If oligo-B is used a sequencing primer, the total length (number of nucleotides) of the SHORTEST DNA chain that would have a dideoxy-G (ddG, 2',3'-dideoxyguanosine) at its end is 5. The DNA chain would be dGTP, dTTP, dATP, dCTP, and finally ddG. When a specific primer is added, the DNA replication starts complementarily from the first primer nucleotide. As per the sequence of oligo-B primer shown, the DNA chain terminating with ddG would be of five nucleotides length.