In reversible terminator sequencing, how would the...

In reversible terminator sequencing, how would the sequencing process be affected if the 3 '-end-blocking group of each nucleotide were replaced with the $3^{\prime}$ - $\mathrm{H}$ present in the dideoxynucleotides used in Sanger sequencing?

Key Concepts

Reversible Terminator Sequencing

This is a sequencing method where nucleotides are modified with temporary 3'-blocking groups that prevent further DNA synthesis once incorporated. The blocking groups are later chemically removed, allowing the controlled, single-base incorporation and detection of fluorescent signals in each cycle. This stepwise process is essential for reading the DNA sequence accurately at the single nucleotide resolution.

Sanger Sequencing

Also known as chain-termination sequencing, Sanger sequencing uses dideoxynucleotides that lack a 3'-OH group. When these nucleotides are incorporated by DNA polymerase, they cause permanent termination of extension, creating a mixture of fragments that can be separated by size to deduce the sequence. This method contrasts with reversible terminator sequencing by irreversibly halting polymerase activity.

3'-end Blocking Groups

In the context of sequencing, the 3'-end blocking groups are chemical modifications attached to nucleotides to temporarily stop DNA polymerase from adding further bases. In reversible terminator sequencing, these groups are crucial as they allow controlled, single-nucleotide incorporation and ensure that only one nucleotide is added per cycle. The later removal of these blocks is what enables continuous sequencing.

Impact of 3'-end Modifications on Polymerase Activity

The presence or absence of a 3'-blocking group determines whether DNA polymerase can continue elongating a DNA strand. When replaced with an unblocked 3'-hydrogen (as in dideoxynucleotides of Sanger sequencing), the polymerase would not be halted in a reversible manner but would cause irreversible termination, fundamentally altering the stepwise control that reversible terminator sequencing relies on. This change would lead to uncontrolled chain extension or permanent termination rather than a controlled, single-base addition.

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