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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Kaiser RJ,MacKellar SL,Vinayak RS,Sanders JZ,Saavedra RA,Hood LEdoi
10.1093/nar/17.15.6087subject
Has Abstractpub_date
1989-08-11 00:00:00pages
6087-102issue
15eissn
0305-1048issn
1362-4962journal_volume
17pub_type
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