Question

Dideoxy nucleotides that are used in DNA sequencing experiments terminate DNA synthesis because they:

		cannot be recognized by DNA polymerases and therefore are not incorporated during synthesis.
		have no 3′ -OH group and so no additional nucleotides can be added after these nucleotides are incorporated.
		have no phosphate groups.
		are only recognized by reverse transcriptases.
		have methyl groups attached to the 5′ position of the sugar and cannot be incorporated during synthesis.

Answer 1

Dideoxy nucleotides have no 3'-OH group and so no additional nucleotides can be added after these nucleotides are incorporated.

The Sanger's method of DNA sequencing is based upon chain termination, which occurs due to the incorporation of dideoxy nucleotides. Sample DNA, that needs to be sequenced, is divided into four tubes. To each of the four tubes, primer sequence, DNA polymerase and standard deoxy nucleotides are added. One additional ingredient added into these tubes is dideoxy nucleotides. Synthesis of new DNA is allowed to take place. New deoxy nucleotides are continued to be added, but as a dideoxy nucleotide is added, extension terminates. The structure of the dideoxy nucleotide is responsible for this termination. Dideoxy nucleotides, unlike deoxy nucleotides, do not have 3'-OH group, on the other hand, phosphodiester bond is created between the 3'-OH of the last incorporated nucleotide and the 5'-phosphate of the incoming nucleotide. In the absence of 3'-OH group, DNA polymerase cannot add new nucleotides. Hence, addition of dideoxy nucleotides terminates DNA synthesis.

The structure of a dideoxy nucleotide has been given below-