Authors: Sutherland T.C.; Dinsmore M.J.; Kraatz H-B.; Lee J.S.

Source: Biochemistry and Cell Biology, Volume 82, Number 3, June 2004 , pp. 407-412(6)

Publisher: NRC Research Press

Abstract:

A 50-base Guide strand was synthesized that consisted of a central 10-base probe sequence flanked by two tracts of 20 adenine residues. Target sequences of 10 bases containing up to three mismatches were prepared and hybridized to the Guide strand in 1 M KCl. The transport of these constructs through single -hemolysin pores was analysed by measuring the current blockade as a function of time. Complementary dsDNA takes significantly longer (840 ± 60 µs) to pass through the pore than a sequence of the same length containing a single (590 ± 45 µs) and a double (270 ± 50 µs) mismatch. Constructs involving three mismatches were indistinguishable from Guide ssDNA transport (120 ± 30 µs). The results suggest that dsDNA must unzip as it is transported through the nanopore. Duplexes containing mismatches unzip more quickly and can be distinguished from those with perfect complementarity.Key words: DNA unzipping, bacterial nanopores, DNA transport, single-molecule detection, DNA mismatch.

Un brin Guide de 50 bases, comprenant une séquence sonde centrale de 10 bases flanquée de deux séquences de 20 résidus adénine, a été synthétisé. Des séquences cibles de 10 bases ayant jusqu'à trois changements de base entraînant un mauvais appariement ont été synthétisées et hybridées au brin Guide en présence de KCl 1 M. Le transport de ces construits au travers de pores d'á-hémolysine a été étudié en mesurant le blocage de courant en fonction du temps. Pour passer au travers d'un pore, l'ADN double brin complémentaire nécessite significativement plus de temps (840 ± 60 µs) qu'une séquence de même longueur ayant un seul (590 ± 45 µs) ou deux mauvais appariements (270 ± 50 µs). Le transport des construits ayant trois mauvais appariements prends le même temps que celui de l'ADN Guide simple brin (120 ± 30 µs). Ces résultats suggèrent que l'ADN double brin doit se séparer au moment de son transport au travers du nanopore. Les duplex comprenant de mauvais appariements se dissocient plus rapidement et peuvent ainsi être distingués de ceux dont la complémentarité est parfaite.Mots clés : séparation de l'ADN, nanopores bactériens, transport de l'ADN, blocage de courant, mauvais appariment de l'ADN.[Traduit par la Rédaction]

Document Type: Research article

Publication date: 2004-06-01

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