Method of eliminating background amplification of nucleic acid targets

Patent metadata

Field	Value
Publication number	US-11111529-B2
Application number	US-201715998749-A
Country	US
Kind code	B2
Filing date	Feb 16, 2017
Priority date	Feb 16, 2016
Publication date	Sep 7, 2021
Grant date	Sep 7, 2021

Abstract

An object is to provide a method of eliminating background amplification in detection of rare nucleic acids based on isothermal amplification. The method comprising steps of: preparing a mixture including solution and enzymes, and adding a first and a second template into the mixture, wherein the first template is an amplification template, and the second template is a leak absorption template. Methods to use this approach for multiplexed, ultrasensitive and ultra-specific detection of nucleic acids are presented.

First claim

The invention claimed is:

1. A method of isothermal amplification of a nucleic acid target sequence to obtain an amplified sequence, the method comprising:
(a) obtaining a mixture of:
(i) buffer comprising a polymerase, nicking enzyme and exonuclease,
(ii) a first oligonucleotide which is an amplification oligonucleotide having a nucleic acid sequence between 10 and 40 nucleotides, wherein the sequence includes a partial repeat structure containing a nicking enzyme recognition site,
(iii) a second oligonucleotide which is a leak absorption oligonucleotide having a nucleic acid sequence between 10 and 35 nucleotides, a 3′ end that is complementary to the amplified sequence, and a 5′ end that encodes a sequence of a tail which is appended to the amplified sequence, the sequence of the tail having a length between 2 and 6 nucleotides and being different from the amplified sequence, and
(iv) a third oligonucleotide which is a target-specific conversion oligonucleotide having a 3′ end that is complementary to the nucleic acid target sequence and a 5′ end at least partially complementary to 3′ end of the amplification oligonucleotide;

(b) adding the mixture of (a) to a sample to be analyzed;
(c) incubating the sample at constant temperature; and
(d) detecting the nucleic acid target sequence through amplification of the amplified sequence, while eliminating background amplification by the leak absorption oligonucleotide.

2. The method according to claim 1 , wherein the first oligonucleotide is able to bind and exponentially amplify the amplified sequence and the second oligonucleotide is able to bind, extend, deactivate and release the products of polymerization along the first oligonucleotide, thereby inducing a threshold, which corresponds to a minimal concentration of the amplified sequence under which the amplification reaction is repressed and above which it is activated.

3. The method according to claim 1 , wherein a 3′ end of the third oligonucleotide binds to a target sequence to obtain, upon polymerization and nicking, a sequence able to activate the first oligonucleotide above the threshold adjusted by controlling concentration of the second oligonucleotide.

4. The method according to claim 1 , wherein the polymerase and the nicking enzyme can drive the isothermal amplification and the exonuclease can avoid saturation of the system.

5. The method according to claim 1 , wherein amplification is initiated only when a mixture of enzymes and oligonucleotides receives stimulation above a predetermined threshold.

6. The method according to claim 2 , wherein the threshold is adjusted by controlling a concentration of the second oligonucleotide.

7. The method according to claim 1 , wherein a 3′ end of the first oligonucleotide has a reduced affinity for an amplified sequence, in comparison to the 5′ end output part of the first oligonucleotide and to the binding part of the second leak-absorption oligonucleotide.

8. The method according to claim 2 , wherein a 3′ end of the second oligonucleotide is complementary to the sequence amplified by the first oligonucleotide, and a 5′ end of the second oligonucleotide serves as a template for the polymerase to add a nucleotide sequence at the 3′ end of the amplified sequence.

9. The method according to claim 6 , wherein concentrations of the first and second oligonucleotides are selected so that a reaction of the first oligonucleotide is faster than a reaction of the second oligonucleotide at suprathreshold concentration of the amplified sequence but the reaction on the second oligonucleotide is faster than the reaction of the first oligonucleotide at subthreshold concentration of the amplified sequence, wherein the threshold is between 10 aM and 20 nM, thereby eliminating amplification unless the stimulus threshold is crossed.

10. The method according to claim 1 , wherein the target sequence is an RNA or DNA strand of known sequence, which can be used as a biomarker.

11. The method according to claim 10 , wherein multiple target sequences are simultaneously detected within the same sample using multiple sets of the first, the second and the third oligonucleotide with orthogonal sequences able to detect and report independently their specific targets.

12. The method according to claim 1 , wherein the method further comprises adding a fourth oligonucleotide which is a reporting probe.

13. The method according to claim 12 , wherein the reporting probe is a fluorescent probe.

14. The method according to claim 12 , wherein the reporting probe detects the amplified sequences amplified by the amplification oligonucleotide.

15. The method according to claim 12 , wherein the reporting probe is a single strand with a self-complementary structure modified at both ends by a fluorophore and/or a quencher.

16. The method according to claim 12 , wherein the reporting probe comprises a loop which includes a nicking recognition site.

17. The method according to claim 1 , wherein the amplification oligonucleotide contains a recognition site of a restriction enzyme.

18. The method according to claim 17 , wherein the degradation speed of the double strand amplification template is controlled by varying the ratio nickase/restriction enzyme.

19. The method according to claim 17 , wherein a restriction enzyme is added.

20. The method according to claim 19 , wherein a restriction enzyme is added with an exonuclease.

Assignees

• Univ Tokyo
• Centre Nat Rech Scient
• The Foundation For The Promotion Of Ind Science

Inventors

• Rondelez Yannick
• Gines Guillaume
• Montagne Kevin
• Fujii Teruo

Classifications

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6848 Primary

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12Q1/6844 Primary

  Nucleic acid amplification reactions · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title