Abstract
The progress of antisense DNA therapy demands development of reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method of phosphorothioate antisense DNA sequencing analysis using UV detection coupled to capillary electrophoresis (CE) has been developed based on a modified chain termination sequencing method. The proposed method reduces the sequencing cost since it uses affordable CE-UV instrumentation and requires no labeling with minimal sample processing before analysis. Cycle sequencing with ThermoSequenase generates quantities of sequencing products that are readily detectable by UV. Discrimination of undesired components from sequencing products in the reaction mixture, previously accomplished by fluorescent or radioactive labeling, is now achieved by bringing concentrations of undesired components below the UV detection range which yields a 'clean', well defined sequence. UV detection coupled with CE offers additional conveniences for sequencing since it can be accomplished with commercially available CE-UV equipment and is readily amenable to automation.
Full Text
The Full Text of this article is available as a PDF (145.9 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Belenky A., Smisek D. L., Cohen A. S. Sequencing of antisense DNA analogues by capillary gel electrophoresis with laser-induced fluorescence detection. J Chromatogr A. 1995 May 12;700(1-2):137–149. doi: 10.1016/0021-9673(94)01092-s. [DOI] [PubMed] [Google Scholar]
- Brownstein M. J., Carpten J. D., Smith J. R. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques. 1996 Jun;20(6):1004-6, 1008-10. doi: 10.2144/96206st01. [DOI] [PubMed] [Google Scholar]
- Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hu G. DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment. DNA Cell Biol. 1993 Oct;12(8):763–770. doi: 10.1089/dna.1993.12.763. [DOI] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tabor S., Richardson C. C. A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6339–6343. doi: 10.1073/pnas.92.14.6339. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tang J., Roskey A. M., Agrawal S. Method for sequencing synthetic oligodeoxynucleotide phosphorothioates. Anal Biochem. 1993 Jul;212(1):134–137. doi: 10.1006/abio.1993.1302. [DOI] [PubMed] [Google Scholar]
- Tessier D. C., Brousseau R., Vernet T. Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase. Anal Biochem. 1986 Oct;158(1):171–178. doi: 10.1016/0003-2697(86)90606-8. [DOI] [PubMed] [Google Scholar]
- Wyrzykiewicz T. K., Cole D. L. Sequencing of oligonucleotide phosphorothioates based on solid-supported desulfurization. Nucleic Acids Res. 1994 Jul 11;22(13):2667–2669. doi: 10.1093/nar/22.13.2667. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zamecnik P. Background of the antisense oligonucleotide approach to chemotherapy. Antisense Nucleic Acid Drug Dev. 1997 Jun;7(3):199–202. doi: 10.1089/oli.1.1997.7.199. [DOI] [PubMed] [Google Scholar]