Method for detecting a circularized DNA, and use of said method for detecting mutations

摘要

The present invention relates to a method for detecting a circularized single-stranded DNA by means of the isothermal hyperbranched rolling circle amplification technique, in which the primers used comprise a detectable barcode sequence and, optionally, a spacer which blocks polymerization by DNA polymerase. The present invention also relates to the use of said method for detecting a genetic polymorphism of one or more base pair(s).

主权项

1. A method for detecting a circularized single-stranded DNA comprising the steps of:
(i) performing hyperbranched rolling circle amplification (HRCA) of said circularized single-stranded DNA in the presence of a forward primer that is capable of hybridizing with said circularized single-stranded DNA and with negative strands generated during the HRCA, and a reverse primer that is capable of hybridizing with positive strands generated during the HRCA, said HRCA generating periodic double-stranded DNAs, wherein:
said forward primer is constituted by or comprises the following sequence, from its 5′ end to its 3′ end: 5′-(F1)n1-T1-(E1)m1-A1-3′, in which:
F1 represents a terminal group selected from a tag and a coupling agent;T1 represents a barcode nucleotide sequence constituted by 6 to 30 nucleotides;E1 represents a spacer that blocks polymerization of the strand complementary to said nucleotide sequence T1 by a DNA polymerase deprived of exonuclease activity and having a strand displacement activity;A1 represents a nucleotide sequence constituted by 10 to 40 nucleotides that is capable of hybridizing with said circularized single-stranded DNA and with said negative strands; andn1 and m1 are independently a whole number equal to 0 or 1; and/orsaid reverse primer is constituted by or comprises the following sequence, from its 5′ end to its 3′ end: 5′-(F2)n2-T2-(E2)m2-A2-3′, in which:
F2 represents a terminal group selected from a tag and a coupling agent, which may be identical to or different from the terminal group F1;T2 represents a barcode nucleotide sequence constituted by 6 to 30 nucleotides, which may be identical to or different from the nucleotide sequence T1;E2 represents a spacer that blocks polymerization of the strand complementary to said nucleotide sequence T2 by a DNA polymerase deprived of exonuclease activity and having a strand displacement activity, and which may be identical to or different from the spacer E1;A2 represents a nucleotide sequence constituted by 10 to 40 nucleotides that is capable of hybridizing with said positive strands; andn2 and m2 are independently a whole number equal to 0 or 1;wherein m1+m2 is equal to 1 or 2; and(ii) detecting said circularized single-stranded DNA by hybridizing said barcode nucleotide sequences T1 and/or T2 present at the ends of said double-stranded periodic nucleic acids with a nucleotide probe complementary to said barcode sequences T1 and/or T2, said nucleotide probes being present on a solid support.