Method and kit for nucleic acid sequence detection
First Claim
1. A method of detecting the presence of a target sequence in a polynucleotide analyte contained in a sample, comprising:
- (a) mixing the sample with a target probe having a sequence capable of hybridizing with said target sequence, under conditions effective to form a double-stranded complex of the analyte and the probe, (b) reacting the probe in the complex in the presence of a polymerase and a selected one to three of four possible nucleotide triphosphates, thereby to add a selected one or more target-directed nucleotide bases to the probe'"'"'s 3′
end to produce a modified probe, (c) hybridizing the modified probe with a detection probe, where the detection probe does not hybridize to the target and at least one of the two probes has a sticky end having a single-stranded portion complementary to the other probe'"'"'s single-stranded portion at its 3′
end or 5′
end. (d) by said reacting ligating the two probes to form a two-probe ligation product, and (e) detecting the presence of the ligation product.
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Abstract
A method and kit for detecting the presence of a target sequence in a polynucleotide analyte contained in a sample are disclosed. In practicing the method, the sample is mixed with a target probe having a sequence capable of hybridizing with said target sequence, under conditions effective to form a double-stranded complex of the analyte and the probe, and the probe in the complex is reacted in the presence of a polymerase and a selected one to three of four possible nucleotide triphosphates, to add a selected one or more target-directed nucleotide bases to the probe'"'"'s 3′ end to produce a modified probe. The modified probe is hybridized with a detection probe, the two probes are ligated to form a two-probe ligation product, and the presence of the ligation product is detected.
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Citations
25 Claims
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1. A method of detecting the presence of a target sequence in a polynucleotide analyte contained in a sample, comprising:
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(a) mixing the sample with a target probe having a sequence capable of hybridizing with said target sequence, under conditions effective to form a double-stranded complex of the analyte and the probe, (b) reacting the probe in the complex in the presence of a polymerase and a selected one to three of four possible nucleotide triphosphates, thereby to add a selected one or more target-directed nucleotide bases to the probe'"'"'s 3′
end to produce a modified probe,(c) hybridizing the modified probe with a detection probe, where the detection probe does not hybridize to the target and at least one of the two probes has a sticky end having a single-stranded portion complementary to the other probe'"'"'s single-stranded portion at its 3′
end or 5′
end.(d) by said reacting ligating the two probes to form a two-probe ligation product, and (e) detecting the presence of the ligation product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A kit for detection of an RNA analyte, such as an RNA viral genome, an siRNA, or an miRNA, having a known target sequence, comprising,
(a) a single-stranded DNA target probe having a sequence capable of hybridizing with said RNA target sequence, under conditions effective to form a double-stranded heteroduplex complex of the analyte and the probe, (b) a reverse transcriptase enzyme, (c) one to three of four possible nucleotide triphosphates, (d) a single-stranded DNA detection probe with a sticky end having a single strand portion that is complementary to a 3′ - -end sequence of the target probe after addition of the selected nucleotide(s) to the 3′
-end of the target probe, where the detection probe (i) does not hybridize to the target and (ii) contains a promoter sequences,(e) a polymerase, and (f) a detection label capable of producing a detectable signal thereby, wherein addition of an RNA analyte and aqueous medium to a reaction vessel, and addition of the kit components, is effective to initiate reactions to (a) form a double-stranded complex of the analyte and target probe, (b) add a selected one or more target-directed nucleotide bases to the target probe'"'"'s 3′
end and a reverse transcriptase to produce modified probe, (c) ligate the modified target probe and the detection probe to form a two-probe ligation product, (d) amplifying ligation-product to produce amplicons containing analyte-target sequences, and (e) bind the detection label to said amplicons, to produce a detectable signal in the vessel. - View Dependent Claims (24, 25)
- -end sequence of the target probe after addition of the selected nucleotide(s) to the 3′
Specification