PRIMERS AND PROBES FOR NUCLEIC ACID DETECTION

摘要

A process for detecting the presence or absence of at least one nucleotide variation in sequence in one or more nucleic acids in a sample comprises: (a) treating the sample, together or sequentially with 4 different nucleotide triphosphates (I), an agent (II) for polymerisation of (I), and one oligonucleotide primer for each strand of each nucleic acid suspected of contg. the variation(s) under hybridising conditions, such that for each strand contg. each different variation, an extension prod. of each primer is sinthesised which is complementary to each strand, where the primer(s) are selected to be complementary to each strand contg. each variation, such that the extension prod. synthesised from one primer, when sepd. from its complement, can serve as a template for synthesis of the extension prod. of the other primer; (b) treating the sample under denaturing conditions to separate primer extension prods. from their templates if the variation(s) are present (c) treating the sample, together or sequentially, with the four (I), (II) and oligonucleotide primers such that primer extension prod. is synthesised using each of the single strands produced in (b), and repeating (b) and (c) to give detectable amplification of the nucleic acid contg. the variation(s), if present; (d) affixing the prod. of (c) to a membrane; (e) treating the membrane under hybridisation conditions with a labelled sequence -specific oligonucleotide probe capable of hybridising with the amplified nucleic acid contg. the variation(s), if present; (d) affixing the prod. of (c) to a membrane; (e) treating the membrane under hybridisation conditions with a labelled sequence-specific oligonucleotide probe capable of hybridising 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 the presence of hybridisation.