Question

1) Why does the addition of dideoxynucleotides to the DNA sequencing reaction cause DNA synthesis to stop after the modified nucleotide is added?
A)Because once a modified nucleotide is incorporated into the DNA, there is no free 5'OH group to attach the next nucleotide being added.
B)Because once a modified nucleotide is incorporated into the DNA, there is no free 3'PO4-group to attach the next nucleotide being added.
C)Because once a modified nucleotide is incorporated into the DNA, there is no free 3'OH group to attach the next nucleotide being added.
D)Because once a modified nucleotide is incorporated into the DNA, there is no free 5'PO4-group to attach the next nucleotide being added.

Answer 1

In Sanger sequencing, once a modified nucleotide is incorporated into the DNA, there is no free 3'OH group to attach the next nucleotide being added. This phenomenon is useful to sequence a polynucleotide chain.

Dideoxynucleotides (ddNTPs) are nucleotide inhibitors of the DNA polymerase, it is for that reason that they are used in Sanger sequencing.

The ddNTPs are similar to natural deoxynucleotides used by cells during ADN replication; however, ddNTPs lack a hydroxyl (OH) group on the 3’ carbon of the deoxyribose.

In consequence, once a ddNTP is added, DNA synthesis cannot progress, thereby this phenomenon can be used to determine the order of nucleotides during the elongation of a polynucleotide DNA chain.

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