Patent ID: 8501459
Filing Date: 2013-08-06
Classification: C12Q

Abstract:
1. A nucleic acid detection method comprising the following steps: (1) preparing a nucleic acid sample; (2) preparing detection probes comprising a detection probe pair (P1 and P2) used for detecting an individual base, wherein each of detection probes P1 and P2 contains either one of two sequences (H1 and H2) respectively complementary to the two flanking sequences of the base site and a relevant common oligonucleotide (H3 or H4), wherein the detection probe pair contains at least a specific Tag sequence, and wherein the base site is in the nucleic acid sample; (3) annealing and hybridizing detection probes P1 and P2 to the nucleic acid sample so that an one nucleotide gap is generated between detection probes P1 and P2 on a hybridization complex formed by detection probes P1 and P2 and the nucleic acid sample, thereby obtaining an annealed reaction system, wherein, when the gap is filled by a nucleotide, the nucleotide filled in the gap exactly corresponds to the base at the base site in the nucleic acid sample; (4) dividing the annealed reaction system obtained by step (3) into four different equal reaction systems of A, T, G and C by adding only dATP to reaction system A, adding only dTTP to reaction system T, adding only dGTP to reaction system G, adding only dCTP to reaction system C; performing four different ligation reactions by adding a DNA polymerase and a DNA ligase into each of the four different equal reaction systems of A, T, G and C; and forming four different ligation mixtures so that the gap is filled by one of dATP, dTTP, dGTP and dCTP and a ligated probe is produced by ligating detection probes P1 and P2; (5) separately purifying the four different ligation mixtures; (6) performing four different amplification reactions using the four different ligation mixtures purified in step (5) and forming four different amplification reaction mixtures, wherein one or two of the four different amplification reaction mixtures have an amplification product comprising the ligated product; (7) preparing a common oligonucleotide chip comprising immobilized different oligonucleotide probes and dividing the chip into four different hybridization regions of A, T, G and C; wherein the oligonucleotide probes in hybridization region of A comprise the specific Tag sequence and only hybridize to the ligated probe when the gap is filled by dATP, the oligonucleotide probes in hybridization region of T comprise the specific Tag sequence and only hybridize to the ligated probe when the gap is filled by dTTP, the oligonucleotide probes in hybridization region of G comprise the specific Tag sequence and only hybridize to the ligated probe when the gap is filled by dGTP, and the oligonucleotide probes in hybridization region of C comprise the specific Tag sequence and only hybridize to the ligated probe when the gap is filled by dCTP; (8) hybridizing each of the four different amplification reaction mixtures to its corresponding hybridization regions of A, T, G, or C on the oligonucleotide chip respectively and producing one or more hybridization signals; and (9) detecting and analyzing the one or more hybridization signals obtained from step (8).