Sensitive and selective nucleic acid detection

摘要

Specific detection of a nucleic acid (A) in a sample comprises (a) reacting the sample with 1 or more labelled mononucleotide triphosphates (I) and one or more enzymes which catalyse prepn. of labelled nucleic acid (B) contg. (I); (b) recting the sample with a nucleic acid probe (C) which is adequately complementary to (B) and (c) detecting the hybrid (D) formed between (B) and (C). The new features are that (1) (C) contains at least one immobilisable gp. (2) after step (a) a non-thermal denaturation is carried out; (3) (D) is contacted with a solid phase which can bind specifically to immunoilisable probe (c); (4) the liq. and solid phases are sepd. and (5) the label is detected on the solid phase. Step (a) is carried out in presence of a specific initiator and the enzymes used produce a (B) essentially complementary to (A). The initiator is pref. a primer (P1) complementary to a a part of (A) and is extended in step (a). A second primer (P2) complementary to part of (B) can also be included and after extension reaction the prods. are sepd., reacted again with P1/P2 and used as template for extension of the corresp. primer.