Polymorphism Detection Method
First Claim
1. A method for detecting the presence of a polymorphism at a particular position within a target nucleic acid sequence in a sample, said method comprisingi) annealing a pair of probes to said nucleic acid sequence, said probes being designed such that they anneal adjacent each other, and only completely anneal to one form of the sequence, in a reaction mixture which is substantially free of ATP;
- ii) ligating together each completely annealed pair of probes in the presence of NAD and a nucleic acid ligase which uses NAD as a substrate, whereby AMP is released;
iii) phosphorylating the released AMP to ATP; and
iv) detecting ATP in the resultant reaction mixture.
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Accused Products
Abstract
A method for detecting the presence or absence of a polymorphism at a particular position within a nucleic acid sequence in a sample, said method comprising i) annealing a pair of probes to said nucleic acid sequence, said probes being designed such that they anneal adjacent each other, and only completely anneal to one form of the sequence, in a reaction mixture which is substantially free of ATP; ii) ligating together any completely annealed pairs of probes in the presence of NAD and a nucleic acid ligase which uses NAD as a substrate/ iii) phosphorylating any AMP released by a ligation event to ATP, and iv) detecting ATP, in particular using a bioluminescent reaction, in the resultant reaction mixture. Kits for conducting this method are also described and claimed.
115 Citations
18 Claims
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1. A method for detecting the presence of a polymorphism at a particular position within a target nucleic acid sequence in a sample, said method comprising
i) annealing a pair of probes to said nucleic acid sequence, said probes being designed such that they anneal adjacent each other, and only completely anneal to one form of the sequence, in a reaction mixture which is substantially free of ATP; -
ii) ligating together each completely annealed pair of probes in the presence of NAD and a nucleic acid ligase which uses NAD as a substrate, whereby AMP is released; iii) phosphorylating the released AMP to ATP; and iv) detecting ATP in the resultant reaction mixture. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 18)
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- 10. A kit for detecting a polymorphism within a target nucleic acid sequence, said kit comprising a nucleic acid ligase which uses NAD as a substrate, a phosphorylating agent, and one or more reagents able to produce a bioluminescent signal.
Specification