US 2022/0205024 A1 · MELTING TEMPERATURE METHODS, KITS AND REPORTER OLIGO FOR DETECTING VARIANT NUCLEIC ACIDS

A method for detecting the presence of a variant sequence in a target nucleic acid sequence consisting of two strands and comprising nucleotide(s) of interest (NOI) preferably comprising repeats, wherein said target nucleic acid sequence consists of a variant sequence or of a reference sequence, said method comprising the steps of:

a) Providing a first sample comprising nucleic acids suspected of comprising said variant sequence;
b) Providing a second sample comprising nucleic acids comprising said reference sequence, wherein the second sample is a reference sample;
c) Providing a reporter oligonucleotide;
d) Providing a set of primers consisting of a first primer and a second primer, wherein the set of primers together are capable of amplifying the target nucleic acid sequence comprising the NOI;
e) Amplifying the target nucleic acid sequence in the presence of said first sample, said first primer and said second primer, thereby obtaining a first amplicon comprising nucleic acids suspected of comprising a variant sequence;
and amplifying the target nucleic acid sequence in the presence of said second sample, said first primer and said second primer, thereby obtaining a second amplicon comprising the reference sequence, wherein the second amplicon is a reference amplicon;
f) Performing melting analysis, such as high-resolution melt (HRM) analysis, of the first amplicon, thereby obtaining a first profile characterised by a first melt curve, and performing melting analysis, such as HRM analysis, of the second amplicon, thereby obtaining a second profile, characterised by a second melt curve wherein the second profile is a reference profile characterised by a reference melt curve;
wherein each amplicon comprises a first strand and a second strand, wherein the melting analysis involves hybridisation of the reporter oligonucleotide to one strand of each amplicon, detection of a signal emitted by the fluorophore, and obtaining the first and the second melt curves;
wherein the reporter oligonucleotide is a sequence of in the range of 10 to 50, preferably in the range of 15 to 50 nucleotides, into which in the range of 2 to 10 hydrophobic nucleotides have been inserted,
wherein the reporter oligonucleotide comprises a first fluorophore, preferably in its 5′-end or within 4 nucleotides from the 5′-end, and a first quencher, preferably in its 3′-end or within 4 nucleotides from the 3′-end, and
wherein the reporter oligonucleotide comprises a hybridization sequence H,
wherein the hybridisation sequence is identical to a consecutive stretch of the sequence of a first strand of the target nucleic acid sequence, and wherein the hybridisation sequence is complementary to a consecutive stretch of the sequence of the second strand of the target nucleic acid sequence,
and wherein the hybridisation sequence of the reporter oligonucleotide comprises or consists of a repetitive sequence and at least one helper sequence in its 5′-end and/or in its 3′-end, wherein said helper sequence does not comprise repeats, and can hybridise to the first and second amplicons when the hybridisation sequence is hybridized thereto;
and
g) Comparing the first profile to the reference profile, wherein a difference between the first profile and the reference profile indicates that the first sample contains a variant sequence.

12 more elements

The method according to any one of the preceding claims, wherein

at least one hydrophobic nucleotide is positioned at the 5′-end or within 10 nucleotides from the 5′-end of the reporter oligonucleotide;
and/or
at least one hydrophobic nucleotide is positioned at the 3′-end or within 10 nucleotides from the 3′-end of the reporter oligonucleotide;
and
wherein the hydrophobic nucleotide has the structure
X—Y-Q
wherein
X is a nucleotide or nucleotide analogue or a backbone monomer unit capable of being incorporated into the backbone of a nucleic acid or nucleic acid analogue,
Q is an intercalator which is not taking part in Watson-Crick hydrogen bonding;
and
Y is a linker moiety linking said nucleotide or nucleotide analogue or backbone monomer unit and said intercalator.

8 more elements

A kit of parts for detecting the presence of a variant sequence in a target nucleic acid sequence consisting of two strands and comprising nucleotide(s) of interest (NOI) preferably comprising repeats, wherein said target nucleic acid sequence consists of a variant sequence or of a reference sequence, said kit of parts comprising:

a) a reporter oligonucleotide comprising a first fluorophore, preferably in its 5′-end or within 4 nucleotides from the 5′-end, and a first quencher, preferably in its 3′-end or within 4 nucleotides from the 3′-end,
wherein the reporter oligonucleotide is a sequence of in the range of 10 to 50 nucleotides, preferably in the range of 15 to 50 nucleotides, into which in the range of 2 to 10 hydrophobic nucleotides have been inserted and
wherein the reporter oligonucleotide comprises a hybridization sequence H, and
wherein the hybridisation sequence is identical to a consecutive stretch of the sequence of a first strand of the target nucleic acid, and wherein the hybridisation sequence is complementary to a consecutive stretch of the sequence of a second strand of the target nucleic acid;
and wherein the hybridisation sequence of the reporter oligonucleotide comprises or consists of a repetitive sequence and at least one helper sequence in its 5′-end and/or in its 3′-end, wherein said helper sequence does not comprise repeats, and can hybridise to the first and second amplicons when the hybridisation sequence is hybridized thereto;
and
b) a set of primers consisting of a first primer and a second primer, wherein the set of primers together are capable of amplifying the target nucleic acid sequence.

4 more elements

A reporter oligonucleotide which can hybridise to one strand of a target nucleic acid consisting of two strands and comprising nucleotide(s) of interest (NOI) preferably comprising repeats, said reporter oligonucleotide comprising a first fluorophore, preferably in its 5′-end or within 4 nucleotides from the 5′-end, and a first quencher, preferably in its 3′-end or within 4 nucleotides from the 3′-end, wherein the reporter oligonucleotide is a sequence of in the range of 10 to 50, preferably in the range of 15 to 50 nucleotides, into which in the range of 2 to 10 hydrophobic nucleotides have been inserted and wherein the reporter oligonucleotide comprises a hybridization sequence H,

wherein the hybridisation sequence is identical to a consecutive stretch of the sequence of a first strand of the target nucleic acid, and wherein the hybridisation sequence is complementary to a consecutive stretch of the sequence of a second strand of the target nucleic acid,
and wherein the hybridisation sequence of the reporter oligonucleotide comprises or consists of a repetitive sequence and at least one helper sequence in its 5′-end and/or in its 3′-end, wherein said helper sequence does not comprise repeats, and can hybridise to the second strand of the first and second amplicons when the hybridisation sequence is hybridized thereto.

Melting temperature methods, kits and reporter oligo for detecting variant nucleic acids

Life of this patent

Melting temperature methods, kits and reporter oligo for detecting variant nucleic acids

Life of this patent

Abstract

Claims26