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High-quality automated DNA sequencing primed with hexamer strings

M A Lodhi¹, W R McCombie

¹Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

Genome Research

|January 1, 1996

Summary

This study introduces a cost-effective method for genome sequencing using short oligonucleotide primers. The new protocol significantly improves primer walking efficiency and read length on automated sequencers.

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Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Genome sequencing finishing is costly due to custom primer synthesis and primer walking.
Short oligonucleotide libraries offer a potential cost-reduction strategy by eliminating custom synthesis.
Previous hexamer string methods showed promise but required optimization for fluorescent sequencing.

Purpose of the Study:

To develop and validate a robust protocol for using hexamer strings in fluorescent DNA sequencing.
To achieve read lengths comparable to traditional dye-terminator sequencing.
To reduce the costs and complexity associated with genome sequencing primer walking.

Main Methods:

Development of a two-cycle fluorescent Sequenase terminator procedure.
Testing of 32 different 3-hexamer primer strings on M13mp18 single-stranded DNA template.
Utilizing ABI 373 DNA sequencers for automated sequencing.

Main Results:

Achieved a 97% success rate for hexamer primer string priming.
Obtained an average read length of 461 bases with >99% base-calling accuracy.
Demonstrated comparable performance to longer primers in dye-terminator sequencing.

Conclusions:

The developed protocol is robust and universally applicable for primer walking on single-stranded templates.
This method significantly reduces costs and increases efficiency in automated DNA sequencing.
The protocol is amenable to automation, further streamlining genome sequencing workflows.

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