METHODS FOR AMPLIFYING NUCLEIC ACID USING TAG-MEDIATED DISPLACEMENT

摘要

Disclosed are methods for amplifying a nucleic acid target region using an amplification oligomer comprising a target-binding segment and a heterologous displacer tag situated 5′ to the target-binding segment. Initiation of an amplification reaction from the tagged amplification oligomer produces an amplicon comprising the displacer tag, such that once the complement of the displacer tag has been incorporated into a second amplicon, a displacer oligonucleotide having a sequence substantially corresponding to the displacer tag sequence is used to participate in subsequent rounds of amplification for displacement of an extension product primed from a site within the second amplicon 5′ to the displacer priming site. Also disclosed are related kits and reaction mixtures comprising the displacer-tagged amplification oligomer and corresponding displacer oligonucleotide.

主权项

1. A method of amplifying a nucleic acid target region, the method comprising:
contacting a target nucleic acid comprising the target region with (1) a first amplification oligomer comprising
(a) a target-binding priming segment (T1) substantially complementary to a 3′-end of the target region;(b) a first heterologous displacer tag (D1) located 5′ to T1; and(c) an intervening spacer segment (S1) between T1 and D1;said contacting comprising conditions whereby the target nucleic acid serves as a template for extension from the first amplification oligomer to produce a first amplification product comprising T1 and D1; (2) a second amplification oligomer comprising a target-binding segment T2 substantially complementary to a region of the first amplicon that is the complement of a 5′-end of the target region, and wherein said contacting further comprises conditions whereby the first amplicon serves as a template to produce a second amplicon comprising segments cT1 and cD1, complementary to T1 and D1, respectively; (3) a third amplification oligomer comprising target-binding priming segment T1p having a nucleotide sequence substantially corresponding to T1, or substantially corresponding to the complement of a second amplicon target sequence cT1′ near or overlapping with cT1 and situated 5′ to cD1; and (4) a fourth amplification oligomer comprising a displacer priming segment D1p having a nucleotide sequence substantially corresponding to D1; wherein said contacting further comprises conditions whereby the second amplicon serves as a template for extension from both the third and fourth amplification oligomers, wherein extension of T1p from a T1p:cT1/cT1′ hybrid produces a third amplicon, and wherein extension of D1p from a D1p:cD1 hybrid produces a fourth amplicon while displacing the third amplicon.