Oligonucleotide Ligation Assay By Detecting Released Pyrophosphate
OwnerNigel Tooke, Bjorn Ekstrominventors
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Abstract
The present invention is related to a new method for determining the presence of genetic element(s), such as nucleotide repeat(s), or marker(s) for microbial typing, in a nucleic acid sample. The method is based on performing a ligation reaction where after a by-product of the ligation is detected and used to determine the number of nucleotidc repeat units in a nucleic acid sample possibly comprising a nucleotide repeat. The invention is also related to kits for performing the method of the present invention and compositions comprising components for performing the present invention.
Claims34
Method for determining the presence of a genetic element such as a nucleotide repeat or a marker for microbial typing in a nucleic acid sample, which method comprises the steps of:
- a) providing a nucleic acid sample comprising a genetic element;
- b) providing an oligonucleotide that is completely or partially complementary to a region comprising the genetic element of said nucleic acid sample;
- c) annealing said oligonucleotide to said nucleic acid sample;
- d) ligating at least two of said oligonucleotides annealed to said nucleic acid sample to each other using a ligase enzyme;
- and
- e) detecting a ligation-by-product to determine whether a ligation reaction has occurred, as a measure of the presence of the genetic element,
- wherein steps a)-e) are performed simultaneously or subsequently or in any combination of subsequent steps.
Method for analysing the number of nucleotide repeats in a nucleic acid sample, which method comprises the steps of:
- a) providing a nucleic acid sample potentially comprising a nucleotide repeat;
- b) providing an oligonucleotide complementary to said nucleotide repeat;
- c) annealing said oligonucleotide to said nucleic acid sample;
- d) ligating at least two of said oligonucleotides annealed to said nucleic acid sample to each other using a ligase enzyme;
- and
- e) detecting a ligation by-product to determine whether a ligation reaction has occured,
- wherein steps a)-e) are performed simultaneously or subsequently or in any combination of subsequent steps.
Method for analysing the number of nucleotide repeats in a nucleic acid sample, which method comprises the steps of:
- a) providing a nucleic acid sample potentially comprising a nucleotide repeat;
- b) providing an oligonucleotide complementary to said nucleotide repeat;
- c) annealing said oligonucleotide to said nucleic acid sample;
- d) ligating at least two of said oligonucleotides annealed to said nucleic acid sample to each other using a ligase enzyme;
- e) converting a ligation by-product into ATP;
- and
- f) detecting said ATP to determine whether a ligation reaction has occured,
- wherein steps a)-f) are performed simultaneously or subsequently or in any combination of subsequent steps.
Method for analysing the number of nucleotide repeats in a nucleic acid sample, which method comprises the steps of:
- a) providing a nucleic acid sample potentially comprising a nucleotide repeat;
- b) providing an oligonucleotide complementary to said nucleotide repeat;
- c) annealing said oligonucleotide to said nucleic acid sample;
- d) ligating at least two of said oligonucleotides annealed to said nucleic acid sample to each other using a ligase enzyme;
- e) converting a ligation by-product into ATP;
- and
- f) detecting said ATP by a luciferase-based assay as a measure of whether a ligation reaction has occured,
- wherein steps a)-f) are performed simultaneously or subsequently or in any combination of subsequent steps.
Method for microbial typing of a nucleic acid sample, which method comprises the steps of:
- a) providing a nucleic acid sample comprising at least one marker for microbial typing;
- b) providing an oligonucleotide that is completely or partially complementary a to region comprising a marker for microbial typing of said nucleic acid sample;
- c) annealing said oligonucleotide to said nucleic acid sample;
- d) ligating at least two of said oligonucleotides annealed to said nucleic acid sample to each other using a ligase enzyme;
- and
- e) detecting a ligation by-product to determine whether a ligation reaction has occurred;
- and
- f) comparing the ligation pattern of the sample with a reference pattern in order to determine the microbial type,
- wherein steps a)-e) are performed simultaneously or subsequently or in any combination of subsequent steps.
Composition comprising a ligase enzyme and an enzyme for converting a ligation by-product into ATP.
Dependencies are inferred from the claim text.