Ola-based methods for the detection of target nucleic avid sequences

摘要

Method for the detection of a target sequence comprising ligating two probes when annealed adjacent to the target sequence, hybridization of a compound primer to the ligated probes and after elongation of the compound primer, amplifying the elongated compound primer from primers annealing to primer binding sites provided in the compound primer and one of the probes to produce detectably amplicons.

主权项

1. A method for determining the presence, absence or amount of a target nucleotide sequence in a nucleic acid sample, the method comprising the steps of:
a) providing to a nucleic acid sample a first probe for each target sequence to be detected in the sample, whereby the first probe has a first target specific section that is complementary to a first part of the target sequence and a second probe for each target sequence to be detected in the sample, whereby the second probe is different from the first probe and has a second target specific section that is complementary to a second part of the target sequence, whereby the first and second part of the target sequence are located adjacent to each other, and whereby the second probe further comprises a tag section that is essentially non-complementary to the target sequence, whereby the tag section comprises a first primer binding sequence; b) allowing the first and second target specific sections of the first and second probe to anneal to the first and second parts of each target sequence that is present in the sample whereby the first and second target specific sections of the probes are annealed adjacent on the target sequence; c) providing means for connecting the first and second target specific sections annealed adjacently to the target sequence and allowing the first and second target specific sections to be connected, to produce a connected probe corresponding to a target sequence in the sample; d) providing to the mixture resulting from step c) a compound primer that is different from both the first probe and the second probe and comprises a section that is complementary to at least part of the first target specific section and further comprises a second primer binding section; e) allowing the compound primer to anneal to at least part of the first target specific section; f) elongating the compound primer; g) providing a set of primers comprising a first primer having a sequence essentially identical to the first primer-binding section, and a second primer that is complementary to the second primer-binding section; h) amplifying the resulting mixture to produce an amplified sample comprising amplicons that are representations of the connected probes; i) determining the presence, absence or amount of a target sequence in the sample by detecting the presence, absence or amount of the corresponding amplicon.