Gene analysis method using SDL-PCR

Patent metadata

Field	Value
Publication number	US-9523117-B2
Application number	US-201214002662-A
Country	US
Kind code	B2
Filing date	Jan 30, 2012
Priority date	Mar 4, 2011
Publication date	Dec 20, 2016
Grant date	Dec 20, 2016

Abstract

The present invention relates to a method for analyzing genes using SDL-PCR (separation of displaced ligation probe-based PCR), and more particularly to a method for analyzing genes using SDL-PCR, in which probes comprising a nucleotide sequence complementary to the gene of interest are ligated with each other by ligase, and another probe capable of hybridizing to the probes is hybridized and extended, thereby preparing a template probe, and the template probe for the gene of interest is amplified using universal primers.
According to the SDL-PCR method of the present invention, non-specific amplification can be minimized by removing non-ligated probes or genomic DNA using a tag, and separation can be achieved within a shorter time compared to a separation method that is performed using exonuclease. In addition, ligation, separation and polymerase chain reaction processes can be performed in a single solution in a single tube, and thus a plurality of genes can be amplified at the same time in an accurate and rapid manner.

First claim

The invention claimed is:

1. An SDL-PCR method for amplifying a template probe for a gene of interest using universal primers, the method comprising the steps of:
(a) mixing a mixture including the gene of interest with a mixture including probe 1 and probe 2, and adding ligase thereto to ligate probe 1 and probe 2 with each other;
(b) adding probe 3, DNA polymerase and dNTP to the mixture resulting from step (b) to hybridize probe 3 to probe 2, and extending probe 3 so as to be complementary to the nucleotide sequences of probe 2 and probe 1, thereby preparing a template probe for the gene of interest;
(c) separating the template probe using a tag; and
(d) amplifying the separated template probe of step (c) by a polymerase chain reaction (PCR) using a primer pair corresponding to a universal forward primer nucleotide sequence and a universal reverse primer nucleotide sequence,
wherein the probe 1 includes, from 5′ direction to 3′ direction, the following: a universal forward primer nucleotide sequence and a nucleotide sequence complementary to a portion of a gene of interest,
wherein the probe 2 includes, from 5′ direction to 3′ direction, the following: a nucleotide sequence complementary to a 5′ to 3′ region following the portion of a gene of interest of probe 1 and an additional nucleotide sequence which hybridizes to probe 3,
wherein the probe 3 includes, from 3′ direction to 5′ direction, the following: a nucleotide sequence complementary to the additional nucleotide sequence of probe 2, a universal reverse primer nucleotide sequence, and a tag for separation,
wherein the universal reverse primer nucleotide sequence and the nucleotide sequence complementary to the additional nucleotide sequence of probe 2 are different,
wherein the tag is selected from the group consisting of biotin, avidin, streptavidin, antigens, antibodies, host compounds, guest compounds, metal chelate compounds, and nucleic acids, and
wherein the primer corresponding to the universal reverse primer nucleotide sequence further comprises a fluorescent substance to determine the presence or absence of an amplification product.

2. The SDL-PCR method of claim 1 , wherein the ligase is selected from the group consisting of E. coli DNA ligase, Taq DNA ligase, and T4 DNA ligase.

3. The SDL-PCR method of claim 1 , wherein the DNA polymerase is selected from the group consisting of Taq DNA polymerase and Pfu DNA polymerase.

4. The SDL-PCR method of claim 1 , wherein the presence or absence of the amplification product is determined using a DNA chip having a capture probe immobilized thereon.

5. An SDL-PCR method for amplifying a template probe group for a plurality of genes of interest using universal primers, the method comprising the steps of:
(a) mixing a mixture including the plurality of genes of interest with a mixture including probe group 1 and probe group 2, and adding ligase thereto to ligate each probe 1 and each probe 2 with each other, which correspond to each of the plurality of genes of interest;
(b) adding probe 3, DNA polymerase and dNTP to the mixture resulting from step (a) to hybridize probe 3 to each probe 2, and extending probe 3 so as to be complementary to the nucleotide sequences of probes 2 and probes 1, thereby preparing a template probe group including template probes for the plurality of genes of interest,
(c) separating the template probe using a tag; and
(d) amplifying the separated template probe of step (c) by a polymerase chain reaction (PCR) using a primer pair corresponding to a universal forward primer nucleotide sequence and a universal reverse primer nucleotide sequence,
wherein the probe group 1 comprises a plurality of probes 1, each of which include from 5′ direction to 3′ direction, the following: a universal forward primer nucleotide sequence and a nucleotide sequence complementary to a portion of a gene of interest,
wherein the probe group 2 comprises a plurality of probes 2, each of which include from 5′ direction to 3′ direction, the following: a nucleotide sequence complementary to a 5′ to 3′ region following the portion of a gene of interest of probe 1 and an additional nucleotide sequence which hybridizes to probe 3,
wherein the probe 3 includes, from 3′ direction to 5′ direction, the following: a nucleotide sequence complementary to the additional nucleotide sequence of probe 2, a universal reverse primer nucleotide sequence, and a tag for separation,
wherein the universal reverse primer nucleotide sequence and the nucleotide sequence complementary to the additional nucleotide sequence of probe 2 are different,
wherein the tag is selected from the group consisting of biotin, avidin, streptavidin, antigens, antibodies, host compounds, guest compounds, metal chelate compounds, and nucleic acids, and
wherein the primer corresponding to the universal reverse primer nucleotide sequence further comprises a fluorescent substance to determine the presence or absence of an amplification product.

6. The SDL-PCR method of claim 5 , wherein the ligase is selected from the group consisting of E. coli DNA ligase, Taq DNA ligase, T4 DNA ligase.

7. The SDL-PCR method of claim 5 , wherein the DNA polymerase is selected from the group consisting of Taq DNA polymerase and Pfu DNA polymerase.

8. The SDL-PCR method of claim 5 , wherein the presence or absence of the amplification product is determined using a DNA chip having a capture probe immobilized thereon.

9. A method for detecting a plurality of genes, the method comprising the steps of:
(a) preparing and amplifying a template probe group for a plurality of genes of interest using the SDL-PCR method of claim 5 ;
(b) modifying the amplification products into single strands; and
(c) measuring the fluorescence of the modified amplification products to detect the presence or absence of the genes of interest.

10. The method of claim 9 , wherein the detection of the genes of interest is performed using a DNA chip having capture probes immobilized thereon.

11. A method for detecting a plurality of genes, the method comprising the steps of:
(a) amplifying a template probe group for a plurality of genes of interest by the SDL-PCR method of claim 8 , in which probe 3 having varying lengths depending on the genes of interest is used; and
(b) analyzing template probes for the genes of interest in the resulting amplification products using a mass spectrometer, and detecting the presence or absence of the genes of interest based on the presence or absence of peaks for the template probes.

12. A method for detecting mutations in a plurality of genes, the method comprising the steps of:
(a) preparing and amplifying a template probe group for a plurality of genes of interest using the SDL-PCR method of claim 5 ;
(b) modifying the resulting amplification products into single strands; and
(c) measuring the fluorescence of the modified amplification products to detect the presence or absence in mutations in the genes of interest.

13. The method of claim 12 , wherein the detection of mutations in the genes of interest is performed using a DNA chip having capture probes immobilized thereon.

Assignees

• Park Hyun Gyu
• Shin Sung Chul
• Kim Gahee
• Kang Byoung-Cheorl
• Korea Advanced Inst Sci & Tech

Inventors

• Park Hyun Gyu
• Shin Sung Chul
• Kim Gahee
• Kang Byoung-Cheorl

Classifications

• C12Q1/6858 Primary

  Allele-specific amplification · CPC title

• C12Q1/6862

  Ligase chain reaction [LCR] · CPC title

• C12Q1/6853 Primary

  using modified primers or templates · CPC title

• C12Q2521/501

  Ligase · CPC title

• C12Q2561/125

  Ligase Detection Reaction [LDR] · CPC title