DNA sequencing fundamentals — In Sanger (chain-termination) sequencing, what is the primary role of the primer?

Introduction / Context:
Sanger sequencing remains a gold standard for accurate determination of DNA sequence. The method requires a short, single-stranded oligonucleotide primer to initiate DNA polymerase extension on a template. Understanding what the primer controls clarifies how read orientation and coverage are designed in sequencing workflows.

Given Data / Assumptions:

• A primer anneals by complementarity to a known region on the template.
• DNA polymerase extends only from a free 3′-OH end provided by the primer.
• Termination arises from incorporation of dideoxynucleotides (ddNTPs), not from the primer itself.

Concept / Approach:
The primer defines the start site and orientation of sequencing because polymerase extends from the primer’s 3′ end along the template. The distribution of fragment lengths in Sanger sequencing is generated by random incorporation of ddNTPs, which lack the 3′-OH and terminate extension; thus, the primer does not specify where the sequence ends nor does it generate different fragment sizes by itself. Therefore, the primer's role is to specify where the sequence begins.

Step-by-Step Solution:

Verification / Alternative check:
Bidirectional sequencing employs forward and reverse primers to begin sequencing from opposite ends, directly demonstrating the primer’s role in defining where reads begin.

Why Other Options Are Wrong:

• Specifies where the sequence ends: termination is dictated by ddNTP incorporation.
• Both (a) and (b): incorrect; the primer sets only the start.
• Generates different sized fragments: produced by ddNTPs, not the primer alone.

Common Pitfalls:
Confusing PCR (where primers define amplicon ends) with Sanger extension; in Sanger sequencing, a single primer defines the start, and ddNTP events define stops.

Final Answer:
specifies where the sequence begins