Nucleic Acids Research, Vol 26, Issue 18 4249-4258, Copyright © 1998 by Oxford University Press
HK Nguyen, E Bonfils, P Auffray, P Costaglioli, P Schmitt, U Asseline, M Durand, JC Maurizot, D Dupret and NT Thuong
Sequencing by the recently reported hybridization technique requires the
formation of DNA duplexes with similar stabilities. In this paper we
describe a new strategy to obtain DNA duplexes with a thermal stability
independent of their AT/GC ratio content. Melting data were acquired on 35
natural and 27 modified duplexes of a given length and of varying base
compositions. Duplexes built with AT and/or G4EtC base pairs exhibit a
thermal stability restrained to a lower range of temperature than that of
the corresponding natural compounds (16 instead of 51 degrees C). The 16
degrees C difference in thermal stability observed between the least stable
and the most stable duplex built with AT and/or G4EtC base pairs is mainly
due to the sequence effect and not to their AT/G4EtC ratio content. Thus N
-4-ethyl-2'- deoxycytidine (d4EtC) hybridizes specifically with natural
deoxyguanosine leading to a G4EtC base pair whose stability is very close
to that of the natural AT base pair. Oligonucleotide probes involving d4EtC
can be easily prepared by chemical synthesis with phosphoramidite
chemistry. Modified DNA targets were successfully amplified by random
priming or PCR techniques using d4EtCTP, dATP, dGTP and dTTP in the
presence of DNA polymerase. This new system might be very useful for DNA
sequencing by hybridization.
ARTICLES
The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content. Implication for DNA sequencing by hybridization
Centre de Biophysique Moleculaire, UPR 4301 CNRS, University of Orleans, Rue Charles Sadron, 45071 Orleans Cedex 02, France and Appligene-Oncor, Parc d'Innovation, Rue de Geiler de Kaysersberg, BP 72, 67402 Illkirch, France.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Lahoud, V. Timoshchuk, A. Lebedev, M. de Vega, M. Salas, K. Arar, Y.-M. Hou, and H. Gamper Enzymatic synthesis of structure-free DNA with pseudo-complementary properties Nucleic Acids Res., June 1, 2008; 36(10): 3409 - 3419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-K. Nguyen and E. M. Southern Minimising the secondary structure of DNA targets by incorporation of a modified deoxynucleoside: implications for nucleic acid analysis by hybridisation Nucleic Acids Res., October 15, 2000; 28(20): 3904 - 3909. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G Hacia and F. S Collins Mutational analysis using oligonucleotide microarrays J. Med. Genet., October 1, 1999; 36(10): 730 - 736. [Abstract] [Full Text]
|
|