US 2011/0218115 A1 · TEST PROBES, COMMON OLIGONUCLEOTIDE CHIPS, NUCLEIC ACID DETECTION METHOD, AND THEIR USES

Abstract

High-throughput detection for the interesting base or the mutation site in the nucleic acid sample can be achieved by the linear test probe pairs P1 and P2. The test probe pairs P1 and P2 respectively comprise either of the flanking complementary sequences which are adjacent to the interesting base or the mutation site in the nucleic acid sample. When the test probe pairs P1, P2 are annealed and hybridized to the nucleic acid sample, a gap will be generated at the interesting base or the mutation site position between the probe pairs and the sample. Divide the annealed hybrid sample into four equal reaction systems to which add dATP, dTTP, dCTP, dGTP, respectively. The test probe pairs P1 and P2 will be ligated into one single probe when adding the complementary nucleotide system under the DNA polymerase or ligase. After purified and amplified, the generated single probes are hybridized to the corresponding area in a common oligonucleotide microarray. The generated single probe will give a signal in the hybrid area, and therefore detect and analyze the hybrid signal to determine the base type or the mutation genotype at the detection position. The invention can be applied to the re-sequencing the target nucleic acid sequence, the detection and analysis for the mutation, insertion, or deletion sites of a known nucleic acid sequence, and the genotyping of the pathogenic microorganism.

Claims16

Dependent claims

1-23. (canceled)

A detection probe, which is characterized in that the detection probe is essentially composed of a detection probe pair (P1, P2) directing to individual sites to be detected, and the detection probes (P1, P2) respectively contain either one of two sequences respectively complementary to the two flanking sequences of the base site to be detected (H1 or H2), and each also contains a relevant common oligonucleotide sequence (H3 or H4), and the probe pair (P1, P2) further contains at least a specific Tag sequence, and said detection probe pair (P1, P2) can be ligated into one single probe by the following steps:

the detection probe pair anneals and hybridizes to a nucleic acid sample to be detected;
a gap is generated between the probes (P1, P2), which corresponds to the base at the base site to be detected, by hybridizing the sequences respectively complementary to the two flanking sequences of the base site to be detected (H1, H2) to the two flanking sequences of the base site to be detected;
the annealed reaction system is divided into four reaction systems of A, T, G and C equally, to which dATP, dTTP, dGTP and dCTP are added respectively;
the base complementary to that at the base site to be detected will fill the gap in the presence of DNA polymerase and DNA ligase, and thus the detection probe pair is ligated into one single probe.

1 more element

A nucleic acid detection method, which is characterized in that the method comprises the following steps:

{circle around (1)} preparation of a nucleic acid sample to be detected;
{circle around (2)} preparation of detection probes, the detection probes are essentially composed of a detection probe pair (P1, P2) directing to an individual site to be detected, and the detection probes (P1, P2) each contains either one of two sequences respectively complementary to the two flanking sequences of the base site to be detected (H1 or H2), a relevant common oligonucleotide (H3 or H4), and the detection probe pair also contains at least a specific Tag sequence;
{circle around (3)} the probe pair of P1 and P2 is allowed to anneal and hybridize to the nucleic acid sample, and a gap is generated between the probe pair that corresponds to the base at base site to be detected;
{circle around (4)} the annealed reaction system is divided into four equal reaction systems of A, T, G and C, to which dATP, dTTP, dGTP and dCTP are added respectively, the base complementary to the base at base site to be detected fills the gap in presence of DNA polymerase and DNA ligase, and thus the detection probe pair is ligated into one single probe;
{circle around (5)} purification of the above ligated probe;
{circle around (6)} amplification of the purified probe;
{circle around (7)} preparation of a common oligonucleotide chip, dividing each chip into four hybridization regions of A, T, G and C, and the oligonucleotide sequences identical with the Tag sequences on the corresponding probes are spotted on individual site of each region;
{circle around (8)} the four hybridization regions of A, T, G and C of the above oligonucleotide chip hybridize to the probes in the four corresponding amplified reaction systems respectively;
and
{circle around (9)} the results detection and analysis of the chip.

7 more elements

Dependencies are inferred from the claim text.

Test probes, common oligonucleotide chips, nucleic acid detection method, and their uses

Life of this patent

Test probes, common oligonucleotide chips, nucleic acid detection method, and their uses

Life of this patent

Abstract

Claims16