METHOD AND REAGENT FOR DETERMINING SPECIFIC NUCLEOTIDE VARIATIONS
First Claim
Patent Images
1. A method for detecting a specific nucleotide variation at a defined site in a target nucleic acid polymer wherein a first nucleotide residue is replaced by a second nucleotide residue, comprising the steps of:
- (a) hybridizing a detectable amount of a target nucleic acid polymer in single-stranded form with an oligonucleotide primer, the detection step primer, comprising a plurality of nucleotide residues, said primer being complementary to the nucleotide sequence of interest in a region disposed toward the 3′
end from the defined site such that when the primer is hybridized to the polymer there are no nucleotide residues between the defined site and the 3′
end of the primer that are identical to the first or second nucleotide residues to be detected;
(b) extending the primer using a polymerizing agent in a mixture comprising one or more nucleoside triphosphates wherein the mixture includes at least one nucleoside triphosphate complementary to either the first or second nucleotide residue which comprises means for detecting the incorporation of the nucleoside triphosphate in a nucleic acid polymer, and optionally one or more chain terminating nucleoside triphosphates; and
(c) detecting the incorporation of the nucleoside triphosphate, whereby the identity of the nucleotide residue at the defined site is determined.
4 Assignments
0 Petitions
Accused Products
Abstract
Detection of variable nucleotide(s) is based on primer extension and incorporation of detectable nucleoside triphosphates. By selecting the detection step primers from the region immediately adjacent to the variable nucleotide, this variation can be detected after incorporation of as few as one nucleoside triphosphate. Labelled nucleoside triphosphates matching the variable nucleotide are added and the incorporation of a label into the detection step primer is measured. The selection of the detection step primer is important to the method according to this invention and is dependent on the nucleotide sequence of interest. The detection step primers are preferably selected so as to span the region immediately toward the 3′ end from the variable nucleotide to be detected. The detection step primers can also be complementary to a sequence beginning several nucleotides removed from the variable nucleotide. The only limitation concerning the position of the detection step primers is that the sequence between the 3′ end of the detection step primer and the variable nucleotide to be detected must not contain a nucleotide residue of the same type as the one to be detected. The detection step primers can equally well be chosen to be complementary to either the coding or non-coding strands of the nucleotide sequence of interest.
39 Citations
39 Claims
-
1. A method for detecting a specific nucleotide variation at a defined site in a target nucleic acid polymer wherein a first nucleotide residue is replaced by a second nucleotide residue, comprising the steps of:
-
(a) hybridizing a detectable amount of a target nucleic acid polymer in single-stranded form with an oligonucleotide primer, the detection step primer, comprising a plurality of nucleotide residues, said primer being complementary to the nucleotide sequence of interest in a region disposed toward the 3′
end from the defined site such that when the primer is hybridized to the polymer there are no nucleotide residues between the defined site and the 3′
end of the primer that are identical to the first or second nucleotide residues to be detected;
(b) extending the primer using a polymerizing agent in a mixture comprising one or more nucleoside triphosphates wherein the mixture includes at least one nucleoside triphosphate complementary to either the first or second nucleotide residue which comprises means for detecting the incorporation of the nucleoside triphosphate in a nucleic acid polymer, and optionally one or more chain terminating nucleoside triphosphates; and
(c) detecting the incorporation of the nucleoside triphosphate, whereby the identity of the nucleotide residue at the defined site is determined. - View Dependent Claims (4, 5, 6, 7, 8, 9, 11)
-
-
2. A method for detecting a plurality of specific nucleotide variations at defined sites in a target nucleic acid polymer wherein at least a first nucleotide residue is replaced by a second nucleotide residue at a first defined site and a third nucleotide residue is replaced by a fourth nucleotide residue at a second defined site, comprising
(a) hybridizing a detectable amount of a target nucleic acid polymer in single-stranded form with a first oligonucleotide primer, the first detection step primer, comprising a plurality of nucleotide residues, said primer being complementary to the nucleotide sequence of interest in a region disposed toward the 3′ - end from the first defined site such that when the primer is hybridized to the polymer there are no nucleotide residues between the first defined site and the 3′
end of the primer that are identical to the first and second nucleotide residues;
(b) extending the first detection step primer using a polymerizing agent in a mixture comprising one or more nucleoside triphosphates wherein the mixture includes at least one nucleoside triphosphate complementary to the first or second nucleotide residue which comprises means for detecting the incorporation of the nucleoside triphosphate in a nucleic acid polymer, and optionally one or more chain terminating nucleoside triphosphates;
(c) detecting the incorporation of the nucleoside triphosphate, whereby the identity of the nucleotide residue at the first defined site is determined;
(d) removing the extended first detection step primer formed in step (c) from the target nucleic acid polymer; and
(e) adding a second detection step primer, said primer being complementary to the nucleotide sequence of interest in a region disposed toward the 3′
end from the second defined site such that when the primer is hybridized to the immobilized polymer there are no nucleotide residues between the second defined site and the 3′
end of the primer that are identical to the third or forth nucleotide residues to be detected. - View Dependent Claims (10)
- end from the first defined site such that when the primer is hybridized to the polymer there are no nucleotide residues between the first defined site and the 3′
-
3. A method of detecting in a patient a predisposition to a genetic disorder resulting from a specific nucleotide variation at a defined site in genetic material of the patient wherein a first nucleotide residue is replaced by a second nucleotide residue, comprising the steps of:
-
(a) obtaining a sample containing a detectable amount of genetic material derived from the patient;
(b) hybridizing the detectable amount of genetic material in a single-stranded form with a first oligonucleotide primer, the first detection step primer, comprising a plurality of nucleotide residues, said primer being complementary to the nucleotide sequence of interest in a region disposed toward the 3′
end from the first defined site such that when the primer is hybridized to the genetic material there are no nucleotide residues between the defined site and the 3′
end of the primer that are identical to the first and second nucleotide residues;
(c) extending the primer using a polymerizing agent in a mixture comprising one or more nucleoside triphosphates complementary to either the first or second nucleotide residue which comprises means for detecting the incorporation of the nucleoside triphosphate in a nucleic acid polymer, and optionally one or more chain terminating nucleoside triphosphates; and
(d) detecting the incorporation of the nucleoside triphosphate, whereby the identity of the nucleotide residue at the defined site, and thus whether the patient has a predisposition for the associated genetic disorder is determined.
-
-
12. A kit for use in determining specific nucleotide variations in a target nucleic acid polymer comprising in packaged combination
(a) at least one amplification primer comprising an oligonucleotide which is complementary to and hybridizes with a portion of the target nucleic acid polymer and which is effective as a primer for enzymatic nucleic acid polymerization and a first attachment moiety; -
(b) at least one detection step primer comprising an oligonucleotide which is complementary to and hybridizes with a portion 3′
to a variable nucleotide of the target nucleic acid polymer; and
optionally;
(c) at least one solid support comprising a solid matrix and at least one attachment site which is capable of immobilizing the oligonucleotide of the amplification probe through the first attachment moiety; and
(d) at least one nucleoside triphosphate containing means for detecting the incorporation of the nucleoside triphosphate in a nucleic acid polymer. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
-
-
23. A reagent for detecting the presence of a point mutation in which a normal nucleic acid residue is replaced by an abnormal nucleic acid residue at a defined site within a gene of interest, comprising an oligonucleotide of sufficient length to act as a primer for an enzyme catalyzed chain extension nucleic acid polymerization reaction, said oligonucleotide primer having a sequence which is complementary to a region of the gene of interest beginning with the nucleotide residue immediately adjacent to and toward the 3′
- end of the gene from the defined site and extending away from the defined site toward the 3′
end of the gene whereby enzyme catalyzed chain extension nucleic acid polymerization will commence by adding a nucleic acid residue complementary to either the normal nucleic residue or the abnormal nucleic acid residue. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
- end of the gene from the defined site and extending away from the defined site toward the 3′
-
34. A method for detecting, at a defined site in the genome of a microorganism, the existence of point mutations leading to altered pathogenicity or resistance to therapy in the microorganisms wherein a first nucleotide residue is replaced by a second nucleotide residue, comprising the steps of:
-
(a) obtaining a sample containing a detectable amount of genetic material derived from the microorganism;
(b) hybridizing the detectable amount of genetic material in a single-stranded form with an oligonucleotide primer, the detection step primer, comprising a plurality of nucleotide residues, said primer being complementary to the nucleotide sequence of interest in a region disposed toward the 3′
end from the defined site such that when the primer is hybridized to the genetic material there are no nucleotide residues between the defined site and the 3′
end of the primer that are identical to the first or second nucleotide residues to be detected;
(c) extending the primer using a polymerizing agent in a mixture comprising one or more nucleoside triphosphates wherein the mixture includes at least one nucleoside triphosphate complementary to either the first or second nucleotide residue which comprises means for detecting the incorporation of the nucleoside triphosphate in a nucleic acid polymer, and optionally one or more chain terminating nucleoside triphosphates; and
(d) detecting the incorporation of the nucleoside triphosphate, whereby the identity of the nucleotide residue at the defined site and thus whether a point mutation has occurred is determined. - View Dependent Claims (35, 36)
-
-
37. A method for detecting cells having a point mutation at a defined site in the genetic material, wherein a first nucleotide residue is replaced by a second nucleotide residue, when said mutated cells are mixed in a cell population with unmutated cells comprising the steps of:
-
(a) obtaining a detectable quantity of genetic material from the cell population while maintaining the ratio of mutated to unmutated cells;
(b) hybridizing the detectable amount of genetic material in a single-stranded form with an oligonucleotide primer, the detection step primer, comprising a plurality of nucleotide residues, said primer being complementary to the nucleotide sequence of interest in a region disposed toward the 3′
end from the defined site such that when the primer is hybridized to the genetic material there are not nucleotide residues between the defined site and the 3′
end of the primer that are identical to the first or second nucleotide residues to be detected;
(c) extending the primer using a polymerizing agent in a mixture comprising one or more nucleoside triphosphates wherein the mixture includes at least one nucleoside triphosphate complementary to either the first or second nucleotide residue which comprises means for detecting the incorporation of the nucleoside triphosphate in a nucleic acid polymer, and optionally one or more chain terminating nucleoside triphosphates; and
(d) detecting the incorporation of the nucleoside triphosphate, whereby the identity of the nucleotide residue at the defined site and thus whether mutated cells are present is determined. - View Dependent Claims (38, 39)
-
Specification
-
- Resources
Litigation Campaign Assessment
Thank you for your request. You will receive a custom alert email when the Litigation Campaign Assessment is available.
×
-
Current AssigneeLaboratory Corporation Of America Holdings
-
Original AssigneeOrchid Cellmark Incorporated (Laboratory Corporation Of America Holdings)
-
InventorsSODERLUND, HANS E., SYVANEN, ANNE-CHRISTINE
-
Granted Patent
-
Time in Patent OfficeDays
-
Field of Search
-
US Class Current435/6
-
CPC Class CodesC12Q 1/6834 Enzymatic or biochemical co...C12Q 1/6869 Methods for sequencingC12Q 1/6886 for cancer immunoassay for ...C12Q 2535/101 Sanger sequencing method, i...C12Q 2535/125 Allele specific primer exte...C12Q 2563/101 radioactivity, e.g. radioac...
