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Human Genome News, January 1993; 4(5)

BNL Researchers Achieve Sequencing Advance

Hexamer Strings Facilitate Primer Walking for DNA Sequencing

Researchers at the Brookhaven National Laboratory (BNL) recently announced a major advance in the primer walking approach to DNA sequencing. They found that saturating a DNA template with a single-stranded binding (SSB) protein allows strings of hexamers (pieces of DNA that are six nucleotides long) to cooperate in priming DNA synthesis. The discovery, reported in the December 11, 1992, issue of Science by Jan Kieleczawa, John J. Dunn, and F. William Studier, promises to accelerate sequencing tenfold while dramatically decreasing costs.

David Galas, Associate Director of the DOE Office of Health and Environmental Research, says the process should enable scientists to fully automate sequencing and "may be the key to gaining the capability needed to fully sequence the human genome and the genomes of other organisms."

"Walking" Down a Template Chain

In enzymatic sequencing by primer walking, a short, known DNA sequence is used with the enzyme DNA polymerase to "prime" or trigger the synthesis of a new DNA strand complementary to a template of unknown sequence. The sequence of about 500 bases on the new chain can be determined with standard gel electrophoresis methods, and a short stretch at the end of the newly determined sequence is then used to design another primer to extend or "walk" down the next 500 bases. Successive primers selected in this way are used to determine an entire unknown DNA sequence.

The authors believe primer walking is the best strategy for sequencing cosmids (clones containing about 40,000 bp of DNA). The preparation of only one DNA sample is needed to sequence an entire cosmid, without the need for subcloning or multiple template preparation. Primer walking offers low redundancy and high accuracy, and sequence assembly is straightforward. Sequencing of problem areas is facilitated by the freedom to select almost any site for priming.

Conventional primer-walking strategies are slow and expensive; a single primer (usually 15 to 20 bases long) can take up to 2 days to prepare and costs about $50. BNL researchers set out to improve the strategy and reduce the cost. The idea was to use shorter, reusable ("generic") primers that could be stored in a library rather than having to synthesize a new primer for each use.

For sequencing, primers must be long enough to pair at only one place in the template DNA; if more than one site is primed, the reactions interfere with each other and no sequence can be read. A typical 18-base primer would pair only once in 69 billion bases of random sequence, so the researchers reasoned that much shorter primers could be useful in sequencing a template such as a cosmid. Different combinations of short primers might also be used to construct longer, more-specific ones.

Selective Priming

While exploring this approach, the investigators discovered that coating the template DNA with SSB protein from Escherichia coli allows highly selective priming by combinations of three or four hexamers. One hexamer would normally prime at many sites on a cosmid template, making sequence impossible to read. The researchers found that adding SSB prevents individual hexamers from priming but stimulates the combinations to prime if they can pair at adjacent sites on the template. Use of appropriate hexamer strings should allow selective priming at almost any unique stretch of 18 nucleotides in a cosmid DNA. Over 500 hexamers and more than 300 hexamer combinations have been tried, with 60 to 90% of the combinations giving readable sequence.

When the DNA is saturated with SSB (about 2.5 mg SSB/1 mg DNA), very strong priming by the 3' hexamer is obtained. Temperature is critical, with 0 C being optimal; at higher temperatures priming becomes much less efficient. The process works on cosmid DNAs, denatured double-stranded DNA, and polymerase chain reaction products.

Primer Library

A complete library of all 4096 possible hexamers could supply the primers needed to sequence the human genome at less than $0.01 per sequencing reaction, the authors noted. Synthesized on a micromole scale, each hexamer preparation could prime thousands of reactions, and libraries of primers could be distributed at reasonable cost.

The BNL laboratory has purchased an entire hexamer library, which investigators are using to explore the limits of the method and to look for rules for selection of the hexamer strings most likely to prime well. The researchers are now using the material to sequence the genome of a virus containing about 40,000 bp and are planning to sequence the 1-Mb genome of the bacterium that causes Lyme disease.

Automating the Process

If primer walking with hexamer strings proves to be reliable and efficient, the entire sequencing process could be automated. From an array of hexamers, templates, and reagents, robots could assemble sequencing reactions and load the products onto gels. Sequence information could flow directly into a computer, perhaps through fluorescent detection.

A computer could assemble the emerging sequence of each template, select the string of hexamers needed for each step of primer walking, and even select primers for resolving ambiguous or difficult regions of the sequence. The goal is to obtain large amounts of accurate sequence data with little need for skilled human intervention.

To eliminate the electrophoresis bottleneck, the investigators envision an instrument using an array of 100 capillary gels to produce more than 100,000 bp of finished sequence per day.

The success of the method will depend on how efficiently and reliably hexamer strings can prime sequencing reactions on a wide variety of templates. If the method works as well as initial results suggest, it should provide a big boost to genome sequencing.

The independent discovery by Levy Ulanovsky's team (Weizman Institute, Israel) of conditions suitable for primer walking with short oligomers will be described in Proceedings of the National Academy of Sciences.

Reported by Denise Casey
HGMIS, Oak Ridge National Laboratory

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