DNA sequencing by synthesis with degenerate primers

Chao Tang^{a, b},
Xiaolong Shi^a,
Xiujie Li^a,
Zuhong Lu^{a
, ,}

a.
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China
b.
The School of Basic Medical Sciences, Southeast University, Nanjing 210096, China

More Information

Corresponding author: E-mail address: zhlu@seu.edu.cn (Zuhong Lu)

摘要

- /
- /
- /
- /
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.
- microarrays /
- DNA sequencing /
- degenerate primer extension /
- SBS /
- DP-SBS

HTML全文

参考文献(10)

[1]	Braslavsky, I., Hebert, B., Kartalov, E. et al. Sequence information can be obtained from single DNA molecules Proc. Natl Acad. Sci. USA, 100 (2003),pp. 3960-3964
[2]	Drmanac, R., Drmanac, S., Baier, J. et al. DNA sequencing by hybridization with arrays of samples or probes Methods Mol. Biol., 170 (2001),pp. 173-179
[3]	Gunderson, K.L., Kruglyak, S., Graige, M.S. et al. Decoding randomly ordered DNA arrays Genome Res, 14 (2004),pp. 870-877
[4]	Guo, Z., Guilfoyle, R.A., Thiel, A.J. et al. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports Nucleic Acids Res., 22 (1994),pp. 5456-5465
[5]	Mitra, R.D., Shendure, J., Olejnik, J. et al. Anal. Biochem., 320 (2003),pp. 55-65
[6]	Pastinent, T., Kurg, A., Metspalu, A. et al. Minisequencing: A specific tool for DNA analysis and diagnostics on oligonucleotide arrays Genome Res, 7 (1997),pp. 606-614
[7]	Ronaghi, M., Uhlen, M., Nyren, P. A sequencing method based on real-time pyrophosphate Science, 281 (1998),pp. 363-365
[8]	Ruparel, H., Bi, L., Li, Z. et al. Design and synthesis of a 3′-O-allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis Proc. Natl. Acad. Sci. USA, 102 (2005),pp. 5932-5937
[9]	Shendure, J., Porreca, G.J., Reppas, N.B. et al. Accurate multiplex polony sequencing of an evolved bacterial genome Science, 309 (2005),pp. 1728-1732
[10]	Xiao, Y., Lubin, A.A., Baker, B.R. et al. Single-step electronic detection of femtomolar DNA by target-induced strand displacement Proc. Natl. Acad. Sci. USA, 103 (2006),pp. 16677-16680