Process for detecting specific nucleotide variations and genetic polymorphisms present in nucleic acids
AssigneeHoffmann-La Roche Inc.
InventorsHenry A. Erlich, Glenn Horn, Randall K. Saiki, Kary B. Mullis
Life of this patent
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Abstract
Single or multiple nucleotide variations in nucleic acid sequence can be detected in nucleic acids by a process whereby the sample suspected of containing the relevant nucleic acid is repeatedly treated with primers, nucleotide triphosphates, and an agent for polymerization of the triphosphates and then denatured, in a process which amplifies the sequence containing the nucleotide variation if it is present. In one embodiment, the sample is spotted on a membrane and treated with a labeled sequence-specific oligonucleotide probe. Hybridization of the probe to the sample is detected by the label on the probe.
Claims32
A process for detecting the presence of a nucleotide variation in sequence in a nucleic acid contained in a sample, which process comprises:
- (a) treating the sample, together or sequentially, with four different nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and two oligonucleotide primers for said nucleic acid containing said variation under hybridizing conditions, such that a primer hybridizes to said nucleic acid and an extension product of the primer is synthesized which is complementary to said nucleotide variation in sequence, wherein said primers are selected such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of the extension product of the other primer;
- (b) treating the sample under denaturing conditions to separate the primer extension products from their templates;
- (c) treating the sample, together or sequentially, with said four nucleoside triphosphates, an agent for polymerization of the nucleoside triphosphates, and oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template, wherein steps (b) and (c) are repeated a sufficient number of times to result in detectable amplification of the nucleic acid containing the sequence variation;
- (d) directly transferring, without gel fractionation, the product of step (c) to a membrane;
- (e) treating the membrane under hybridization conditions with a labeled sequence-specific oligonucleotide probe between about 15 and about 25 nucleotides in length capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence;
- and
- (f) detecting whether the probe has hybridized to an amplified sequence in the nucleic acid sample.
A kit for detecting the presence of a nucleotide variation in sequence in a nucleic acid contained in a sample, which kit comprises, in packaged form, a multicontainer unit having:
- (a) two oligonucleotide primers for each nucleic acid sequence variation being detected, which primers are selected such that an extension product synthesized from one primer, when separated from its complement, can serve as a template for the synthesis of the extension product of the other primer so as to produce amplified nucleic acid sequences containing the sequence variation;
- (b) an agent for polymerization;
- (c) four different nucleotide triphosphates;
- (d) a sequence-specific oligonucleotide probe between about 15 and about 25 nucleotides in length capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence;
- and
- (e) a membrane to which amplified nucleic acid can bind for subsequent detection by probe hybridization.
A kit for detecting the presence of a nucleotide variation in sequence in a nucleic acid contained in a sample, which kit comprises, in packaged form, a multicontainer unit having:
- (a) two oligonucleotide primers for each nucleic acid sequence variation being detected, which primers are selected such that an extension product synthesized from one primer, when separated from its complement, can serve as a template for the synthesis of the extension product of the other primer so as to produce amplified nucleic acid sequences containing the sequence variation;
- and
- (b) a sequence-specific oligonucleotide probe about 15 to about 25 nucleotides in length, wherein said probe is capable of hybridizing with the amplified nucleic acid sequence only if a sequence of the probe is complementary to a region of the amplified sequence.
Dependencies are inferred from the claim text.