Method for detecting variation of gene for non-diagnostic purpose based on fluorescence quenching and probe thereof

• US 20140272978A1
• Filed: 10/17/2012
• Published: 09/18/2014
• Est. Priority Date: 10/19/2011
• Status: Active Grant

• Current Assignee: Shanghai QY Biotech CP Limited
• Original Assignee: Shanghai QY Biotech CP Limited
• Inventors: Shi, Rui, Huang, Bochun
• Granted Patent: US 9,574,227 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

A method for detecting variation of gene based on fluorescence quenching quantification comprises: performing single base extension at a specific site of a gene to be detected with marked probes and marked dideoxyribonucleotide triphosphate; detecting fluorescence quenching values, and determining SNP of the gene to be detected. The present invention also provides an oligonucleotide probe for the method.

Claims

1-10. -10. (canceled)

11. A method for detecting variation of gene for non-diagnostic purpose based on fluorescence quenching, comprising steps of:

1) extracting DNA from a sample;

2) amplifying a gene segment to be detected based on the DNA;

3) performing an extension reaction with product of the step

     2) as a template, probes, and ddNTP, and performing a fluorescence quenching reaction at the same time;

wherein the probe is marked with quenching molecules, and the ddNTP is marked with fluorescent molecules;

alternatively, the probe is marked with a fluorescence molecule, and the ddNTP is marked with a quenching molecule;

4) detecting changes of fluorescence caused by the extension reaction in the step

     3), and calculating fluorescence quenching values; and

5) determining SNP of gene to be detected according to calculating results in the step

     4).

12. The method, as recited in claim 11, wherein a number for cycles of the extension reaction in the step 3) is 35˜

40.

13. The method, as recited in claim 11, wherein a distance between the fluorescence molecule or the quenching molecule and a sequence 3′

end of the probe marked with the fluorescence molecule or the quenching molecule is 3˜

35 bp.

14. The method, as recited in claim 11, wherein the quenching molecule is BHQ1, BHQ2, BHQ3, TAMRA, Dabcyl or Eclipse, and the fluorescence molecule is fluorescein, R6G, Cy3, Cy5, FAM or VIC.

15. The method, as recited in claim 13, wherein the quenching molecule is BHQ1, BHQ2, BHQ3, TAMRA, Dabcyl or Eclipse, and the fluorescence molecule is fluorescein, R6G, Cy3, Cy5, FAM or VIC.

16. The method, as recited in claim 11, wherein the step 4) further comprises a step of correcting the fluorescence quenching value by an external standard method.

17. The method, as recited in claim 11, wherein the step 4) further comprises steps of calculating fluorescence quenching ratios and quenching determining coefficients, the fluorescence quenching ratio is a ratio of a fluorescence quenching value to an initial fluorescence value, and the quenching determining coefficient is a ratio of the fluorescence quenching ratio of a corresponding channel to a sum of the fluorescence quenching ratios of all the channels.

18. The method, as recited in claim 16, wherein the step 4) further comprises steps of calculating fluorescence quenching ratios and quenching determining coefficients, the fluorescence quenching ratio is a ratio of a fluorescence quenching value to an initial fluorescence value, and the quenching determining coefficient is a ratio of the fluorescence quenching ratio of a corresponding channel to a sum of the fluorescence quenching ratios of all the channels.

19. The method, as recited in claim 18;

wherein in the step

     5), the SNP of the gene to be detected is determined according to the quenching determining coefficient;

if the quenching determining coefficient is not less than 65%, a type of a base is homozygote;

if the quenching determining coefficient is more than 35%, and less than 65%, the type of the base is heterozygote.

20. An oligonucleotide probe for the method, as recited in claim 11;

wherein said oligonucleotide probe comprises at least one probe;

each probe is an oligonucleotide comprising 15˜

45 nucleotides and marked with a fluorescence molecule or a quenching molecule;

a distance between said fluorescence molecule or said quenching molecule and 3′

end of said oligonucleotide sequence marked with said fluorescence molecule or said quenching molecule is 3˜

35 bp.

21. The oligonucleotide probe, as recited in claim 20, wherein said oligonucleotide probe comprises a single probe having a sequence as SEQ ID NO:

3 or SEQ ID NO;

6.

22. The oligonucleotide probe, as recited in claim 20, wherein said oligonucleotide probe comprises double probes respectively having sequences as SEQ ID NO:

7 or SEQ ID NO;

8.