Quantification of nucleic acids

Patent metadata

Field	Value
Publication number	US-9587267-B2
Application number	US-201013255696-A
Country	US
Kind code	B2
Filing date	Mar 9, 2010
Priority date	Mar 10, 2009
Publication date	Mar 7, 2017
Grant date	Mar 7, 2017

Abstract

The invention relates to a method for the quantification of one or more nucleic acids in a sample, for example: making a sample available which contains at least one nucleic acid to be quantified, adding an oligonucleotide probe, the oligonucleotide probe comprising a sequence which can specifically hybridize to the nucleic acid to be quantified or to a common sequence of the nucleic acids to be quantified, incubating the sample under conditions which allow the hybridization of the oligonucleotide probe to the nucleic acid(s) to be quantified, incubating the sample under conditions which allow the extension of hybridized probes, the nucleic acid(s) serving as a template in each case, removing the non-hybridized probes from the sample and quantifying the hybridized oligonucleotide probes to measure the quantity of the nucleic acid(s) to be quantified. The invention also relates to a kit for carrying out said method.

First claim

The invention claimed is:

1. A method for quantifying nucleic acid in a sample, comprising:
(a) providing a sample comprising nucleic acid to be quantified;
(b) adding a sufficient amount of a single-stranded oligonucleotide probe having a length of about 70 to about 100 nucleotides to the sample, thereby forming a probe-nucleic acid sample, the single-stranded oligonucleotide probe comprising a hybridization sequence and a 5′ tag sequence, wherein the hybridization sequence can specifically hybridize to the nucleic acid to be quantified with at least 90% complementarity, and wherein the 5′ tag sequence is located 5′ of the hybridization sequence;
(c) incubating the probe-nucleic acid sample from step (b) under conditions which allow hybridization of the hybridization sequence of the single-stranded oligonucleotide probe to the nucleic acid to be quantified, thereby forming a hybridized nucleic acid sample;
(d) incubating the hybridized nucleic acid sample from step (c) under conditions which allow extension of the single-stranded oligonucleotide probe hybridized to the nucleic acid to be quantified, thereby forming an extended oligonucleotide probe;
(e) after step (d), removing any of the single-stranded oligonucleotide probe added in step (b) and not hybridized to the nucleic acid to be quantified in step (c); and
(f) following step (e), measuring an amount of the nucleic acid to be quantified in the sample by quantifying the extended oligonucleotide probe, wherein said quantifying the extended oligonucleotide probe comprises amplifying the 5′ tag sequence of the extended oligonucleotide probe using a primer that can hybridize to the 5′ tag sequence.

2. The method as claimed in claim 1 , wherein the extension of the single-stranded oligonucleotide probe hybridized to the nucleic acid to be quantified is carried out using a polymerase.

3. The method as claimed in claim 1 , wherein step (e) is performed using a nuclease which can specifically degrade single-stranded nucleic acids.

4. The method as claimed in claim 3 , wherein the nuclease is an exonuclease.

5. The method as claimed in claim 4 , wherein the exonuclease is selected from exonuclease 1, S1 nuclease, lambda exonuclease, and exonuclease VII.

6. The method as claimed in claim 1 , wherein the oligonucleotide probe is selected from DNA, RNA, and locked nucleic acid (LNA).

7. The method as claimed in claim 6 , wherein the oligonucleotide probe contains modified nucleobases and/or modified linkers.

8. The method as claimed in claim 1 , wherein the nucleic acid comprises at least two specific nucleic acids, further comprising adding at least two specific oligonucleotide probes which specifically hybridize to the at least two specific nucleic acids and contain different hybridization sequences and tag sequences to the sample, and quantifying the amount of each of the at least two specific nucleic acids in the sample using steps (c) to (f) of claim 1 , and calculating a ratio of the at least two specific nucleic acids in the sample based on the amount of each of the at least two specific nucleic acids in the sample.

9. The method as claimed in claim 1 , wherein the nucleic acid to be quantified is RNA or DNA.

10. The method as claimed in claim 9 , wherein the RNA is mRNA and the DNA is cDNA.

11. The method as claimed in claim 10 , wherein the nucleic acid to be quantified is mRNA having a common sequence consisting of a poly(A) sequence.

12. The method as claimed in claim 10 , wherein the nucleic acid to be quantified is cDNA having a common sequence consisting of a poly(dT) sequence.

13. The method as claimed in claim 11 , wherein the oligonucleotide probe contains a poly(T) or poly(dT) sequence.

14. The method as claimed in claim 12 , wherein the oligonucleotide probe contains a poly(A) or poly(dA) sequence.

15. The method as claimed in claim 1 , wherein said quantifying the extended oligonucleotide probe is carried out by quantitative PCR.

16. The method as claimed in claim 15 , wherein the nucleic acid to be quantified is mRNA, and an oligonucleotide probe is used as a primer for reverse transcription of the mRNA into cDNA using RT-PCR.

17. The method as claimed in claim 1 , wherein the oligonucleotide probe contains a fluorescent dye.

18. The method of claim 1 , wherein the nucleic acid to be quantified is cDNA, and wherein a common sequence of the cDNA is a poly(dT) sequence, and the hybridization sequence of the single-stranded oligonucleotide probe comprises a poly (A) sequence or a poly(dA) sequence.

19. The method of claim 18 , wherein said quantifying the extended oligonucleotide probe in step (f) is carried out by quantitative PCR.

20. The method of claim 1 , wherein the least 90% complementarity is more than 95% complementarity.

21. A method for quantifying nucleic acid in a sample, comprising:
(a) providing a sample comprising nucleic acid to be quantified, wherein the nucleic acid to be quantified is mRNA or cDNA;
(b) adding a sufficient amount of a single-stranded oligonucleotide probe having a length of about 70 to about 100 nucleotides to the sample, thereby forming a probe-mRNA or probe-cDNA sample, the single-stranded oligonucleotide probe comprising a hybridization sequence and a 5′ tag sequence, wherein the hybridization sequence can specifically hybridize to a common sequence of the mRNA or a common sequence of the cDNA with at least 90% complementarity, and wherein the 5′ tag sequence is located 5′ of the hybridization sequence;
(c) incubating the probe-mRNA or probe-cDNA sample from step (b) under conditions which allow hybridization of the hybridization sequence of the single-stranded oligonucleotide probe to the mRNA or the cDNA, thereby forming a hybridized mRNA or cDNA sample;
(d) incubating the hybridized mRNA or cDNA sample from step (c) under conditions which allow extension of the single-stranded oligonucleotide probe hybridized to the mRNA or the cDNA, thereby forming an extended oligonucleotide probe;
(e) after step (d), removing any of the single-stranded oligonucleotide probe added in step (b) and not hybridized to the mRNA or cDNA in step (c); and
(f) following step (e), measuring an amount of the mRNA or cDNA in the sample by quantifying the extended oligonucleotide probe, wherein said quantifying the extended oligonucleotide probe comprises amplifying the 5′ tag sequence of the extended oligonucleotide probe using a primer that can hybridize to the 5′ tag sequence.

22. The method of claim 21 , wherein the nucleic acid to be quantified is mRNA, and wherein the common sequence of the mRNA is a poly(A) sequence, and the hybridization sequence of the single-stranded oligonucleotide probe comprises a poly(T) sequence or a poly(dT) sequence.

23. The method of claim 22 , wherein said quantifying the extended oligonucleotide probe in step (f) is carried out by quantitative PCR.

24. The method of claim 22 , wherein the extension of the single-stranded oligonucleotide probe hybridized to the mRNA or the cDNA in step (d) is carried out with an enzyme having reverse transcriptase activity.

25. The method of claim 21 , wherein the least 90% complementarity is more than 95% complementarity.

Assignees

• Fang Nan
• Missel Andreas
• Löffert Dirk
• Qiagen Gmbh

Inventors

• Fang Nan
• Missel Andreas
• Löffert Dirk

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2521/325

  Single stranded exonuclease · CPC title