title: DNA sequencing by synthesis with degenerate primers.
abstract: The degenerate primer-based sequencing was developed by a synthesis method (DP-SBS) for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays. In this method, a different set of degenerate primers containing a given number (n) of degenerate nucleotides at the 3'-ends were annealed to the sequenced templates that were immobilized on the solid surface. The nucleotides (n+1) on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides. The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately. The main advantage of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension. From the present study, it is found that the DP-SBS method is reliable, simple, and cost-effective for laboratory-sequencing a large amount of short DNA fragments.
authors.affiliation: State Key Laboratory of Bioelectronics, Southeast University, Nanjing, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Tang
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Shi
authors.forename: Xiujie
authors.initials: X
authors.lastname: Li
authors.forename: Zuhong
authors.initials: Z
authors.lastname: Lu
mesh_terms: D020137:Base Pair Mismatch; D003365:Costs and Cost Analysis; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics*; D054306:Dideoxynucleotides / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006361:Heating; D006878:Hydroxides / Q000737:chemistry; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*; D013696:Temperature; D013997:Time Factors
chemical_list: D017931:DNA Primers; D054306:Dideoxynucleotides; D005456:Fluorescent Dyes; D006878:Hydroxides; D004247:DNA; C031356:hydroxide ion