Detection of mutations in genes by specific LNA primers
First Claim
1. A method for detecting in a sample the presence of a nucleic acid Q′
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, the method comprising the steps of a) combining nucleic acids present in the sample with an appropriate amount of nucleoside triphosphates, an agent for polymerisation of the nucleoside triphosphates and at least one diagnostic oligonucleotide containing at position A a nucleotide which is capable of hybridizing to the nucleotide at position A of nucleic acid Q′
but not to a nucleotide at position A of nucleic acid Q under hybridisation conditions, the at least one diagnostic oligonucleotide containing at least one LNA, b) extending any oligonucleotides which hybridize to the nucleic acids present in the sample to form extension products, wherein said nucleic acids are used as templates, c) detecting any nucleic acids formed in step b) and thereby the presence of nucleic acid Q′
in the sample.
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Abstract
The present invention relates to a method of detecting variant nucleic acid whose nucleotide sequence differs from one another at a single (or more) position(s). The method uses a set of chimeric oligonucleotides containing DNA monomers and monomers of a novel class of DNA analogues, locked nucleic acid (LNA). LNA oligomers obey the Watson-Crick base-pairing rules and form duplexes that are significantly more stable than similar duplexes formed by DNA. The “allele-specific” LNA-containing oligonucleotides wherein the LNA nucleotide(s) are found at the 3′ position can be extended by means of enzymes only where the nucleotide(s), which is/are terminal in direction of extension, is/are complementary to the corresponding nucleotides of the nucleic acid (of the one allele) to be detected. Thus discrimination between alleles without subsequent differential hybridization with labelled oligonucleotides is possible. The invention further relates to reagents for performing the methods as well as applications of the method.
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Citations
53 Claims
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1. A method for detecting in a sample the presence of a nucleic acid Q′
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, the method comprising the steps of
a) combining nucleic acids present in the sample with an appropriate amount of nucleoside triphosphates, an agent for polymerisation of the nucleoside triphosphates and at least one diagnostic oligonucleotide containing at position A a nucleotide which is capable of hybridizing to the nucleotide at position A of nucleic acid Q′
but not to a nucleotide at position A of nucleic acid Q under hybridisation conditions, the at least one diagnostic oligonucleotide containing at least one LNA,b) extending any oligonucleotides which hybridize to the nucleic acids present in the sample to form extension products, wherein said nucleic acids are used as templates, c) detecting any nucleic acids formed in step b) and thereby the presence of nucleic acid Q′
in the sample. - View Dependent Claims (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 48)
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, the method comprising the steps of
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2. A method for detecting in a sample the presence of a nucleic acid Q′
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, the method comprising the steps of
a) combining nucleic acids present in the sample with an appropriate amount of nucleoside triphosphates, an agent for polymerisation of the nucleoside triphosphates and at least one diagnostic oligonucleotide containing at position A a nucleotide which is capable of hybridizing to the nucleotide at position A of nucleic acid Q′
but not to a nucleotide at position A of nucleic acid Q under hybridisation conditions, the at least one diagnostic oligonucleotide containing at least one LNA,b) extending any oligonucleotides which hybridize to the nucleic acids present in the sample to form extension products, wherein said nucleic acids are used as templates, c) after the formation of extension products treating the reaction mixture under denaturing conditions to separate the extension products from the template, d) hybridising in the presence of an appropriate amount of nucleoside triphosphates and an agent for polymerisation of the nucleoside triphosphates the single stranded nucleic acids of step c) with at least one diagnostic oligonucleotide containing at position A a nucleotide which is capable of hybridizing to the nucleotide at position A of nucleic acid Q′
but not to a nucleotide at position A of nucleic acid Q under hybridisation conditions, the at least one diagnostic oligonucleotide containing at least one LNA,e) repeating steps c) and d) a sufficient number of times to result in a detectable amount of extension products, f) detecting the extension products formed.
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, the method comprising the steps of
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3. A method for detecting in a sample the presence of a nucleic acid Q′
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, the method comprising the steps of
a) combining the nucleic acids with an appropriate amount of nucleoside triphosphates, an agent for polymerisation of the nucleoside triphosphates, at least one downstream oligonucleotide and at least one diagnostic oligonucleotide containing at position A a nucleotide which is capable of hybridizing to the nucleotide at position A of nucleic acid Q′
but not to a nucleotide at position A of nucleic acid Q under hybridisation conditions, the at least one diagnostic oligonucleotide containing at least one LNA,b) extending any oligonucleotides which hybridize to the nucleic acids to form extension products, wherein said nucleic acids are used as templates, c) after the formation of extension products treating the reaction mixture under denaturing conditions to separate the extension products from the template, d) hybridising in the presence of an appropriate amount of nucleoside triphosphates and an agent for polymerisation of the nucleoside triphosphates the single stranded nucleic acids from step c) with at least one downstream oligonucleotide and at least one diagnostic oligonucleotide containing at position A a nucleotide which is capable of hybridizing to the nucleotide at position A of nucleic acid Q′
but not to a nucleotide at position A of nucleic acid Q under hybridisation conditions, the at least one diagnostic oligonucleotide containing at least one LNA,e) repeating steps c) and d) a sufficient number of times to result in a detectable amount of extension products, f) detecting the extension products formed.
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, the method comprising the steps of
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4. A method for detecting target nucleic acids whose nucleotide sequences differ from one another in at least one position A, the method comprising the steps of
a) combining the nucleic acids with an appropriate amount of nucleoside triphosphates, an agent for polymerisation of the nucleoside triphosphates and at least one set of diagnostic oligonucleotides under hybridisation conditions, the at least one set of diagnostic oligonucleotides having nucleotide sequences which differ from one another in at least one position A and contain at least one LNA, b) extending any oligonucleotides which hybridize to the nucleic acids to form extension products, wherein said nucleic acids are used as templates, c) detecting the nucleic acids formed in step b).
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5. A method for detecting target nucleic acids whose nucleotide sequences differ from one another in at least one position A, the method comprising the steps of
a) combining the nucleic acids with an appropriate amount of nucleoside triphosphates, an agent for polymerisation of the nucleoside triphosphates and at least one set of diagnostic oligonucleotides under hybridisation conditions, the at least one set of diagnostic oligonucleotides having nucleotide sequences which differ from one another in at least one position A and contain at least one LNA, b) extending any oligonucleotides which hybridize to the nucleic acids to form extension products, wherein said nucleic acids are used as templates, c) after the formation of extension products treating the reaction mixture under denaturing conditions to separate the extension products from the template, d) hybridising in the presence of an appropriate amount of nucleoside triphosphates and an agent for polymerisation of the nucleoside triphosphates the single stranded nucleic acids from step c) with at least one set of diagnostic oligonucleotides whose nucleotide sequences differ from one another in at least one position A to synthesise further extension products, e) repeating steps c) and d) a sufficient number of times to result in a detectable amount of extension products, f) detecting the extension products formed.
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6. A method for detecting target nucleic acids whose nucleotide sequences differ from one another in at least one position A, the method comprising the steps of
a) combining the nucleic acids with an appropriate amount of nucleoside triphosphates, an agent for polymerisation of the nucleoside triphosphates, at least one downstream oligonucleotide and at least one set of diagnostic oligonucleotides under hybridisation conditions, the at least one set of diagnostic oligonucleotides having nucleotide sequences which differ from one another in at least one position A and contain at least one LNA, b) extending any oligonucleotides which hybridize to the nucleic acids to form extension products, wherein said nucleic acids are used as templates, c) after the formation of extension products treating the reaction mixture under denaturing conditions to separate the extension products from the template, d) hybridising in the presence of an appropriate amount of nucleoside triphosphates and an agent for polymerisation of the nucleoside triphosphates the single stranded nucleic acids step c) with at least one downstream oligonucleotide and at least one set of diagnostic oligonucleotides being oligonucleotides whose nucleotide sequences differ from one another in at least one position A to synthesise further extension products, e) repeating steps c) and d) a sufficient number of times to result in a detectable amount of extension products, f) detecting the extension products formed.
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7. A method for detecting target nucleic acids whose nucleotide sequences differ from one another in at least one position A, the method comprising the steps of
a) combining the target nucleic acids with an appropriate amount of nucleoside triphosphates, at least two oligonucleotides wherein at least one of said oligonucleotides is a diagnostic oligonucleotide and an agent for ligation of the oligonucleotides under hybridisation conditions, the at least one diagnostic oligonucleotide being an oligonucleotide containing at position A a nucleotide which is complementary to the nucleotide found at position A of the target nucleic acid to be detected, said diagnostic oligonucleotide contains at least one LNA, b) ligating any oligonucleotides which hybridize to the nucleic acids at adjacent positions to form ligation products, wherein said nucleic acids are used as templates, c) detecting the nucleic acids formed in step b).
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8. A method for detecting target nucleic acids whose nucleotide sequences differ from one another in at least one position A, the method comprising the steps of
a) combining the target nucleic acids with an appropriate amount of nucleoside triphosphates, at least two oligonucleotides wherein at least one of said oligonucleotides is a diagnostic oligonucleotide and an agent for ligation of the oligonucleotides under hybridisation conditions, the at least one diagnostic oligonucleotide being an oligonucleotide containing at position A a nucleotide which is complementary to the nucleotide found at position A of the target nucleic acid to be detected, said diagnostic oligonucleotide contains at least one LNA, b) ligating any oligonucleotides which hybridize to the nucleic acids at adjacent positions to form ligation products, wherein said nucleic acids are used as templates, c) treating the reaction mixture under denaturing conditions to separate the ligation products from the template after the ligation, d) hybridising in the presence of an appropriate amount of nucleoside triphosphates and an agent for ligation of the oligonucleotides under hybridisation conditions the single stranded nucleic acids from step c) with at least one oligonucleotide being complementary to the ligation product from step b) and at least two oligonucleotides the at least one diagnostic oligonucleotide being an oligonucleotide containing at position A a nucleotide which is complementary to the nucleotide found at position A of the target nucleic acid to be detected, said diagnostic oligonucleotide contains at least one LNA, e) repeating steps c) and d) a sufficient number of times to result in a detectable amount of ligation products, f) detecting the ligation products formed.
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49. A kit for detecting in a sample the presence of a nucleic acid Q′
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, comprising
a) an appropriate amount of nucleoside triphosphates, b) an agent for polymerisation of the nucleoside triphosphates, c) at least one diagnostic oligonucleotide containing at position A a nucleotide which is capable of hybridizing to the nucleotide at position A of nucleic acid Q′
but not to a nucleotide at position A of nucleic acid Q under hybridisation conditions, the at least one diagnostic oligonucleotide containing at least one LNA
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, comprising
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50. A kit for detecting in a sample the presence of a nucleic acid Q′
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, comprising
a) an appropriate amount of nucleoside triphosphates, b) an agent for polymerisation of the nucleoside triphosphates, c) at least one downstream oligonucleotide, d) at least one diagnostic oligonucleotide containing at position A a nucleotide which is capable of hybridizing to the nucleotide at position A of nucleic acid Q′
but not to a nucleotide at position A of nucleic acid Q under hybridisation conditions, the at least one diagnostic oligonucleotide containing at least one LNA.
- whose nucleotide sequence differs from a nucleic acid Q in at least one position A, comprising
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51. A kit for detecting target nucleic acids whose nucleotide sequences differ from one another in at least one position A, comprising
a) an appropriate amount of nucleoside triphosphates, b) an agent for polymerisation of the nucleoside triphosphates, c) at least one set of diagnostic oligonucleotides having nucleotide sequences which differ from one another in at least one position A and contain at least one LNA.
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52. A kit for detecting target nucleic acids whose nucleotide sequences differ from one another in at least one position A, comprising
a) an appropriate amount of nucleoside triphosphates, b) an agent for polymerisation of the nucleoside triphosphates, c) at least one downstream oligonucleotide, d) at least one set of diagnostic oligonucleotides having nucleotide sequences which differ from one another in at least one position A and contain at least one LNA.
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53. A kit for detecting target nucleic acids whose nucleotide sequences differ from one another in at least one position A, the kit comprising
a) nucleoside triphosphates, b) an agent for ligation, c) at least two oligonucleotides the at least one diagnostic oligonucleotide being an oligonucleotide containing at position A a nucleotide which is complementary to the nucleotide found at position A of the target nucleic-acid to be detected, said diagnostic oligonucleotide contains at least one LNA.
Specification