Nucleic Acids Research, Vol 24, Issue 15 2998-3004, Copyright © 1996 by Oxford University Press
S Parinov, V Barsky, G Yershov, E Kirillov, E Timofeev, A Belgovskiy and A Mirzabekov
The efficiency of sequencing by hybridization to an oligonucleotide
microchip grows with an increase in the number and in the length of the
oligonucleotides; however, such increases raise enormously the complexity
of the microchip and decrease the accuracy of hybridization. We have been
developing the technique of contiguous stacking hybridization (CSH) to
circumvent these shortcomings. Stacking interactions between adjacent bases
of two oligonucleotides stabilize their contiguous duplex with DNA. The use
of such stacking increases the effective length of microchip
oligonucleotides, enhances sequencing accuracy and allows the sequencing of
longer DNA. The effects of mismatches, base composition, length and other
factors on the stacking are evaluated. Contiguous stacking hybridization of
DNA with immobilized 8mers and one or two 5mers labeled with two different
fluorescent dyes increases the effective length of sequencing
oligonucleotides from 8 to 13 and 18 bases, respectively. The incorporation
of all four bases or 5-nitroindole as a universal base into different
positions of the 5mers permitted a decrease in the number of additional
rounds of hybridization. Contiguous stacking hybridization appears to be a
promising approach to significantly increasing the efficiency of sequencing
by hybridization.
ARTICLES
DNA sequencing by hybridization to microchip octa-and decanucleotides extended by stacked pentanucleotides
Joint Human Genome Program, Engelhardt Institute of Molecular Biology, Moscow, Russia.
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