Patent ID: 8652784
Filing Date: 2014-02-18
Classification: C12Q

Abstract:
1. A method for determining the presence or absence of an insertion polynucleotide at a junction site in a target polynucleotide sequence, wherein the junction site in the target polynucleotide sequence is spanned by a first target sequence on one side and a second target sequence on the other side, the first and second target sequences being contiguous when the insertion polynucleotide is absent, the method comprising: contacting the target polynucleotide sequence with a first primer, a second primer, a blocker polynucleotide, a polymerase, and one or more nucleotide triphosphates and performing an amplification reaction, wherein: (i) the blocker polynucleotide hybridizes to both contiguous first and second target sequences when the insertion polynucleotide is absent, (ii) the first primer hybridizes to a first region of the first target sequence independent of the presence of the insertion polynucleotide, the first region being proximal to the junction site, and (iii) the second primer hybridizes to a second region of the first target sequence, the second region being distal to the junction site, wherein the second primer is capable of priming synthesis of a portion of the first target sequence spanned by the first and second regions of the first target sequence, such that when the insertion polynucleotide is present at the junction site, the first and second primers support exponential amplification of the portion of the first target sequence spanned by the first and second regions of the first target sequence, and when the insertion polynucleotide is absent from the junction site, the blocker polynucleotide hybridizes to the contiguous first and second target sequences to block hybridization of the first primer to the first region so that amplification of the portion of the first target sequence spanned by the first and second regions of the first target sequence is inhibited, whereby the presence or absence of the insertion polynucleotide at the junction site in the target polynucleotide sequence is determined by the presence or absence of amplified portion of the first target sequence spanned by the first and second regions.