Specific-primer-directed DNA sequencing using automated fluorescence detection

Abstract

Automated fluorescence-based DNA sequence analysis offers the possibility to undertake very large scale sequencing projects. Directed strategies, such as the specific-primer-directed sequencing approach ('gene walking'), should prove useful in such projects. Described herein is a study involving the use of this approach in conjunction with automated fluorescence detection on a commercial instrument (ABI 370A DNA sequencer). This includes procedures for the rapid chemical synthesis and purification of labeled primers, the design of primer sequences that are compatible with the commercial analysis software, and automated DNA sequence analysis using such primers. A set of four fluorophore-labeled primers can be reliably synthesized in a twenty-four hour period, and greater than 300 nucleotides of analyzed new sequence obtained using this set in an additional twenty-four hours. Scale-up of these procedures to take advantage of the full capabilities of the sequencer is, at present, too slow and costly to be suitable for routine sequencing, and therefore the use of specific-primers is best suited to the closure of gaps in extended sequence produced using random cloning and sequencing strategies.

Incorporation of Reporter-Labeled Nucleotides by DNA Polymerases

“…These applications have historically relied on radioactive nucleotide substrates, however, there has been increasing emphasis on techniques that use either fluorescent or affinity-based groups such as Biotin or Digoxigenin coupled with enzymatic signal amplification (7). The ability of DNA polymerases to incorporate these modified and labeled nucleotides has been important in monitoring gene expression (8), in situ hybridization (9), and four-color DNA sequencing (10). The methods for introducing nonradioactive detectable groups comprise both enzymatic and chemical additions of 5 and 3 markers, as well as introducing internal markers by enzymatic incorporation or post-labeling techniques (1114).…”

A Personal Journey of Discovery: Developing Technology and Changing Biology

“…Although these simple ideas emerged in a single afternoon, it took another three years before the practical details were solved and a prototype, capillary-based automated DNA sequencing instrument was developed (28). We had to develop a chemistry for coupling the dyes to DNA (29), identify four good dyes, design laser instrumentation for reading dye space, generate algorithms for converting dye space into DNA sequence, optimize the enzymology of polymerases for the extension sequencing reactions, and solve a host of other chemical and engineering challenges. Lloyd Smith played a central role in solving many of these problems.…”

A Quantitative Tool for Producing DNA-Based Diagnostic Arrays

“…The nucleoside couples to the sites that have been exposed to light and, after several steps, an array of ODNs is formed. 3-Tributylstannyl cyanoethylphosphoramidite (Sn-CEP; [26][27][28]), synthesized by ChemGenes Corporation (Waltham, MA; structure shown in Figure 2), was attached at the last step so the amount of Sn should be an accurate measure of the full n-mer at the site (the phosphoramidite will not bind to sites that have been capped off). The signal just off the ends of the area where the ODNs were constructed had a large background of Sn.…”