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. 1997 Aug 1;25(15):3059–3065. doi: 10.1093/nar/25.15.3059

Modification of DNA duplexes to smooth their thermal stability independently of their base content for DNA sequencing by hybridization.

H K Nguyen 1, P Auffray 1, U Asseline 1, D Dupret 1, N T Thuong 1
PMCID: PMC146866  PMID: 9224605

Abstract

The possibility of equalizing DNA duplex stability is essential for the application of sequencing by hybridization. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their base content. Modified *C bases have been developed and incorporated into oligonucleotides. The influence of these modifications on duplex stability has been studied by absorption spectroscopy, thus allowing selection of N -4-ethyl-2'-deoxycytidine (d4EtC), which hybridizes specifically with natural dG to give a G4EtC base pair whose stability is very close to that of natural AT base pairs. Duplexes built with AT and/or G4EtC base pairs exhibit thermal stabilities independent of their base content in a classical buffer solution, thus enabling control of the stability of DNA hybrids as a function of their length only.

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Selected References

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  1. Butkus V., Klimasauskas S., Petrauskiene L., Maneliene Z., Janulaitis A., Minchenkova L. E., Schyolkina A. K. Synthesis and physical characterization of DNA fragments containing N4-methylcytosine and 5-methylcytosine. Nucleic Acids Res. 1987 Oct 26;15(20):8467–8478. doi: 10.1093/nar/15.20.8467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Drmanac R., Labat I., Brukner I., Crkvenjakov R. Sequencing of megabase plus DNA by hybridization: theory of the method. Genomics. 1989 Feb;4(2):114–128. doi: 10.1016/0888-7543(89)90290-5. [DOI] [PubMed] [Google Scholar]
  3. Ehrlich M., Norris K. F., Wang R. Y., Kuo K. C., Gehrke C. W. DNA cytosine methylation and heat-induced deamination. Biosci Rep. 1986 Apr;6(4):387–393. doi: 10.1007/BF01116426. [DOI] [PubMed] [Google Scholar]
  4. Gildea B., McLaughlin L. W. The synthesis of 2-pyrimidinone nucleosides and their incorporation into oligodeoxynucleotides. Nucleic Acids Res. 1989 Mar 25;17(6):2261–2281. doi: 10.1093/nar/17.6.2261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hoheisel J. D., Lehrach H. Quantitative measurements on the duplex stability of 2,6-diaminopurine and 5-chloro-uracil nucleotides using enzymatically synthesized oligomers. FEBS Lett. 1990 Nov 12;274(1-2):103–106. doi: 10.1016/0014-5793(90)81340-t. [DOI] [PubMed] [Google Scholar]
  6. Hoheisel J. D. Sequence-independent and linear variation of oligonucleotide DNA binding stabilities. Nucleic Acids Res. 1996 Feb 1;24(3):430–432. doi: 10.1093/nar/24.3.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jacobs K. A., Rudersdorf R., Neill S. D., Dougherty J. P., Brown E. L., Fritsch E. F. The thermal stability of oligonucleotide duplexes is sequence independent in tetraalkylammonium salt solutions: application to identifying recombinant DNA clones. Nucleic Acids Res. 1988 May 25;16(10):4637–4650. doi: 10.1093/nar/16.10.4637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Khrapko K. R., Lysov YuP, Khorlin A. A., Ivanov I. B., Yershov G. M., Vasilenko S. K., Florentiev V. L., Mirzabekov A. D. A method for DNA sequencing by hybridization with oligonucleotide matrix. DNA Seq. 1991;1(6):375–388. doi: 10.3109/10425179109020793. [DOI] [PubMed] [Google Scholar]
  9. Li S., Haces A., Stupar L., Gebeyehu G., Pless R. C. Elimination of band compression in sequencing gels by the use of N4-methyl-2'-deoxycytidine 5'-triphosphate. Nucleic Acids Res. 1993 Jun 11;21(11):2709–2714. doi: 10.1093/nar/21.11.2709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Maskos U., Southern E. M. A study of oligonucleotide reassociation using large arrays of oligonucleotides synthesised on a glass support. Nucleic Acids Res. 1993 Oct 11;21(20):4663–4669. doi: 10.1093/nar/21.20.4663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Melchior W. B., Jr, Von Hippel P. H. Alteration of the relative stability of dA-dT and dG-dC base pairs in DNA. Proc Natl Acad Sci U S A. 1973 Feb;70(2):298–302. doi: 10.1073/pnas.70.2.298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mikita T., Beardsley G. P. Effects of arabinosylcytosine-substituted DNA on DNA/RNA hybrid stability and transcription by T7 RNA polymerase. Biochemistry. 1994 Aug 9;33(31):9195–9208. doi: 10.1021/bi00197a023. [DOI] [PubMed] [Google Scholar]
  13. Riccelli P. V., Benight A. S. Tetramethylammonium does not universally neutralize sequence dependent DNA stability. Nucleic Acids Res. 1993 Aug 11;21(16):3785–3788. doi: 10.1093/nar/21.16.3785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sung W. L. Synthesis of 4-triazolopyrimidinone nucleotide and its application in synthesis of 5-methylcytosine-containing oligodeoxyribonucleotides. Nucleic Acids Res. 1981 Nov 25;9(22):6139–6151. doi: 10.1093/nar/9.22.6139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Williams J. C., Case-Green S. C., Mir K. U., Southern E. M. Studies of oligonucleotide interactions by hybridisation to arrays: the influence of dangling ends on duplex yield. Nucleic Acids Res. 1994 Apr 25;22(8):1365–1367. doi: 10.1093/nar/22.8.1365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wood W. I., Gitschier J., Lasky L. A., Lawn R. M. Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1585–1588. doi: 10.1073/pnas.82.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]

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