High-quality automated DNA sequencing primed with hexamer strings

Lodhi, M. A., McCombie, W. R. (1996) High-quality automated DNA sequencing primed with hexamer strings. Genome Research, 6 (1). pp. 10-18. ISSN 1054-9803

URL: http://www.ncbi.nlm.nih.gov/pubmed/8681134
DOI: 10.1101/gr.6.1.10

Abstract

The finishing phase of genome sequencing projects is expensive, in part, because of the cost of de novo synthesis of custom primers and the management burden associated with obtaining and using them for primer walking. One approach to reduce these high costs is the use of a presynthesized library of short oligonucleotides (8-10 bases) rather than long primers. The use of such a library eliminates the need for custom synthesis of oligonucleotides, providing the convenience of priming from ally site by combining two to three short oligonucleotides to form a string with the required specificity. The First practical implementation of this strategy presented a robust protocol for using hexamer strings with radioisotopic labeling. Whereas versions of this technique have subsequently been implemented on fluorescent sequencers we felt that there was a need to develop and extensively test a protocol that consistently gave read lengths comparable to dye-terminator sequencing with longer primers. We have developed a new two-cycle Fluorescent Sequenase terminator procedure for using hexamer strings. We tested this procedure using a set of 32 different 3 hexamer primer strings, each known to be Functional to some degree in radioisotopic sequencing, on single-stranded M13mp18 template and ABI 373 DNA sequencers. The overall success rate of priming with these hexamer primer strings is 97% with the Failure of only one string. In this case, the corresponding 18-mer primer also failed to produce usable sequence from M13mp18 template. The average read length from reactions successfully primed with the 31 different hexamer strings was 461 bases with >99% base-calling accuracy. The current protocol is robust enough to be used in virtually any situation where primer walking on single-stranded templates is used. The success rate and read lengths make it universally applicable to the sequencing of single-stranded templates on automated sequencers. It is also amenable to automation.

Item Type: Paper
Uncontrolled Keywords: library walking
Subjects: bioinformatics > genomics and proteomics > analysis and processing
bioinformatics
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics
Investigative techniques and equipment
CSHL Authors:
Communities: CSHL labs > McCombie lab
Depositing User: Matt Covey
Date Deposited: 25 Apr 2013 16:51
Last Modified: 25 Apr 2013 16:51
Related URLs:
URI: http://repository.cshl.edu/id/eprint/28222

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving