Question

In automated DNA sequencing reaction by the Sanger method, which of the following is NOT required?

fluorescently-labeled dideoxynucleotides

deoxynucleotides

DNA polymerase

a DNA primer

none of the above (i.e. all the above are required in the reaction)

Answer 1

None of the above

DNA sequencing is the process for the determination of the order of nucleotides in DNA. Sanger Sequencing or dideoxy sequencing is the most common method used in DNA sequencing which is developed by Frederick Sanger in 1977. This is sequencing by synthesis method. In this process, the DNA to be sequenced is mixed with a radiolabeled primer, Deoxy nucleotides, DNA polymerase enzyme, and an appropriate buffer. Then this aliquoted into 4 separate tubes and each tube is added with one of the dideoxynucleotides. Then this is subjected to in-vitro amplification or PCR.

Didieoxynucleotide is a nucleotide that lacks 3' OH group and due to this, they cannot form a phosphodiester bond. Hence if a dideoxynucleotide incorporated into a DNA during synthesis, the chain termination will occur. As a result, each of the tubes will have a number of fragments with different lengths, and the terminal base of the fragments will be the dideoxynucleotide present in the sample. By separating the DNA fragments through agarose gel electrophoresis and autoradiography, we can deduce the sequence of DNA.

In automated sequencing instead of radiolabeled primers, the fluorescent-labeled dideoxynucleotides are being used. Since the basic principle of the automated sequencing is similar to the conventional Sanger sequencing, it requires other components like DNA polymerase, without this, the amplification will not occur, and Deoxynucleotides, which are the building block of DNA. Since different fluorescent dyes are available, each dideoxynucleotide can be labeled with different dyes and the reaction can be carried out in a single tube. Since it is an automated process, capillary electrophoresis is being used for the separation of DNA fragments and the fragments are detecting using the fluorescent detectors.