Method of nucleic acid fragment detection

• US 10,460,828 B2
• Filed: 09/25/2017
• Issued: 10/29/2019
• Est. Priority Date: 09/30/2016
• Status: Active Grant

• Current Assignee: LifeOS Genomics Corporation
• Original Assignee: LifeOS Genomics Corporation
• Inventors: Liu, Timothy Z., Huang, Ching-Jou
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/714,401
• Publication Number:
  US 20180096098A1
• Time in Patent Office: 764 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2521/113 Telomerase
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16B 40/10 Signal processing, e.g. from mass spectrometry [MS] or from PCR

Abstract

A method of nucleic acid fragment detection includes capturing a target nucleic acid fragment by an oligonucleotide probe to form a hybridised double strand. The oligonucleotide probe has an identification sequence complementary to the target nucleic acid fragment and a reproducible sequence. The hybridised double strand is removed to expose the reproducible sequence of the oligonucleotide probe. The repeats of the reproducible sequence are produced. The repeats of the reproducible sequence are labelled by a detection probe for identification and quantitation.

Claims

1. A method of nucleic acid fragment detection, the method comprising:

capturing a target nucleic acid fragment by an oligonucleotide probe to form a section of hybridised double strand, the oligonucleotide probe having an identification sequence and a reproducible sequence, the identification sequence being complementary to the target nucleic acid fragment;

removing the hybridised double strand section to expose the reproducible sequence of the oligonucleotide probe, wherein the removing the hybridised double strand comprises;

cleaving the hybridized double strand off the oligonucleotide probe by duplex specific nuclease (DSN);

producing repeats of the reproducible sequence; and

labelling the repeats of the reproducible sequence by a detection probe.

2. The method of claim 1, wherein the production of repeats of the reproducible sequence comprises:

adding telomerase.

3. The method of claim 1, wherein the production of repeats of the said reproducible sequence comprises:

adding circularized extension template that comprises a sequence complementary to the reproducible sequence.

4. The method of claim 1, wherein the detection probe comprises a signal label that generates detectable signals, including fluorescence signals, electrochemical signals, or chemiluminescence signals.

5. The method of claim 1, wherein the oligonucleotide probe is fixed on a substrate.

6. The method of claim 5, further comprising:

identifying the target nucleic acid fragment according to a spatial resolution.

7. The method of claim 1, further comprising using a substrate having a plurality of wells, and each of the wells having a plurality of said oligonucleotide probe.

8. The method of claim 7, further comprising:

quantifying a number of the target nucleic acid fragment by counting a number of wells having the presence of the said detection probe.

9. The method of claim 1, further comprising:

providing a substrate having an immobile probe; and

capturing the repeats of the reproducible sequence through the immobile probe, wherein the immobile probe having a sequence complementary to a segment of the oligonucleotide probe.

10. The method of claim 1, further comprising:

removing unbound nucleic acid fragment.

11. The method of claim 1, wherein the target nucleic acid fragment comprises micro ribonucleic acid (miRNA).

12. The method of claim 1, wherein the oligonucleotide probe comprises deoxyribonucleic acid (DNA).

13. The method of claim 1, wherein the reproducible sequence of the oligonucleotide probe comprises an identification tag specific to the target nucleic acid fragment.

14. The method of claim 1, wherein the oligonucleotide probe is fixed on a substrate, the substrate has a plurality of wells, a number of copies of the target nucleic acid fragment is plural, and on average less than one copy of the target nucleic acid fragment is in each of the wells.