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DNA sequencing by primer walking with strings of contiguous hexamers
J Kieleczawa1, J J Dunn, F W Studier
1Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
Science (New York, N.Y.)
|December 11, 1992
Summary
This study shows that DNA binding protein-coated DNA hexamer strings can prime DNA synthesis. This method enables rapid and cost-effective DNA sequencing, potentially increasing efficiency by tenfold.
Area of Science:
- Molecular Biology
- Biochemistry
- Genomics
Background:
- Single-stranded DNA binding proteins (SSBs) are crucial for DNA replication and repair.
- DNA primer selection is a critical step in DNA synthesis.
Purpose of the Study:
- To investigate a novel DNA priming strategy using hexanucleotide (hexamer) strings.
- To assess the feasibility of using hexamer strings for DNA sequencing.
Main Methods:
- Template DNA saturation with SSBs.
- Utilizing cooperative base-stacking interactions of hexamer strings for priming.
- Generating sequence ladders from various DNA templates.
Main Results:
- Hexamers in strings of 3-4 specifically prime DNA synthesis from the 3' end.
- Priming by individual hexamers is suppressed under these conditions.
- Over 200 unique hexamer strings successfully primed readable sequence ladders at >75 sites across diverse DNA templates.
- High success rates (60-90%) were achieved on templates ranging from 6.4 kb to 40 kbp.
Conclusions:
- Hexamers in specific strings offer a robust and specific method for DNA priming.
- Libraries of hexamers can be synthesized affordably for widespread use in sequencing.
- This strategy promises to significantly accelerate and economize large-scale DNA sequencing efforts.
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