# US 11,993,813 B2 · Nucleic acid probe and nucleic acid sequencing method

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,993,813 B2
- Document ID: US11993813B2
- Status: Granted
- Kind: B2
- Application number: 16/643,161
- Filed: 2018-07-06
- Published: 2020-09-03
- Granted: 2024-05-28
- Earliest claimed: 2017-08-31
- Assignees: MGI Tech Co., Ltd.
- Applicants: Mgi Tech Co., Ltd.
- Inventors: Erkai Liu; Wenwei Zhang; Ao Chen; Chongjun Xu
- Web page: https://patentgrep.com/patent/US11993813B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C12Q 1/68: involving nucleic acids
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6853: using modified primers or templates
- C12Q 1/6855: Ligating adaptors
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)

## Abstract

A nucleic acid probe and a nucleic acid sequencing method for performing sequencing while ligating nucleic acids. The nucleic acid probe is a DNA sequencing probe, comprising a first moiety, a second moiety, a linker, and a detectable label. A base of the first moiety is A, T, U, C, or G, a base of the second moiety is a random base and/or a universal base, and 3 bases or more are present in the second moiety. The first moiety and the second moiety are ligated via the linker, the connection between the first moiety and the ligation can be cleaved, and the detectable label is ligated to the second moiety or the linker. The above probe, a combination formed therewith, or a sequencing method using the same can reduce the number or types of probes in nucleic acid sequencing, thereby reducing cost.

## Claims

### Claim 1

A nucleic acid probe combination, which comprises 4 groups of nucleic acid probes, wherein each group of nucleic acid probes contains a nucleic acid probe, wherein the nucleic acid probe comprises a first moiety, a second moiety, a linker and a detectable label, wherein:
the first moiety has a base of A, T, U, C or G,
the second moiety has random bases and/or universal bases, and the number of the bases is 3 or more,
the first moiety is ligated to the second moiety via the linker, and the ligation between the first moiety and the linker can be cleaved,
the detectable label is ligated to the second moiety or the linker; and
the linker does not contain a sulfur atom;
wherein:
the first group of nucleic acid probes: comprising the nucleic acid probe of which the base of the first moiety is A;
the second group of nucleic acid probes: comprising the nucleic acid probe of which the base of the first moiety is T or U;
the third group of nucleic acid probes: comprising the nucleic acid probes of which the base of the first moiety is C;
the fourth group of nucleic acid probes: comprising the nucleic acid probes of which the base of the first moiety is G;
and the detectable labels in the 4 groups of nucleic acid probes are different from each other;
the 4 groups of nucleic acid probes are mixed or not mixed;
the mole number of the first group of nucleic acid probes and that of the fourth group of nucleic acid probes are equal;
the mole number of the second group of nucleic acid probes and that of the third group of nucleic acid probes are equal;
the sum of the mole number of the first group of nucleic acid probes and that of the fourth group of nucleic acid probes is less than or equal to the sum of the mole number of the second group of nucleic acid probes and that of the third group of nucleic acid probes; and
the molar ratio of the first group of nucleic acid probes:the second group of nucleic acid probes:the third group of nucleic acid probes:the fourth group of nucleic acid probes is (0.5-2):(3-5):(3-5):(0.5-2); more preferably 1:4:4:1.

### Claim 2 (depends on 1)

A kit, which comprises the nucleic acid probe combination according to claim 1;
preferably, the kit further comprises one or more selected from the group consisting of a reagent capable of cleaving the ligation between the first moiety and the linker, a buffer for dissolving the nucleic acid probe, and a sequencing primer;
preferably, the reagents in the kit are free of silver ion.

### Claim 3 (depends on 2)

The kit according to claim 2, wherein the reagent capable of cleaving the ligation between the first moiety and the linker is an endonuclease, an organic phosphide, or a complex of PdCl2 and sulfonated triphenylphosphine.

### Claim 4 (depends on 1)

A ligation solution, which comprises the nucleic acid probe combination according to claim 1, and a DNA ligase.

### Claim 5 (depends on 4)

The ligation solution according to claim 4, characterized in any one or more of the following (1) to (5):
(1) the DNA ligase is one or more selected from the group consisting of T4 DNA ligase, T7 DNA ligase, and T3 DNA ligase;
(2) the concentration of the nucleic acid probe is 0.1 μM to 5 μM, preferably 1 μM;
(3) the concentration of the DNA ligase is 0.01 μM to 2 μM, preferably 0.5 μM;
(4) further comprising the following components: 50 mM CH3COOK, 20 mM Tris, 10 mM Mg(CH3COO)2, 100 μg/ml BSA, 1 mM ATP, 10% PEG6000;
(5) the rest of the ligation liquid is water.

### Claim 6 (depends on 1)

A method for sequencing nucleic acid, comprising the following steps:
(1) hybridizing a sequencing primer to a nucleic acid molecule to be tested;
(2) ligating the nucleic acid probe combination according to claim 1 to the sequencing primer;
(3) eluting the nucleic acid probe that has not bound to the nucleic acid molecule to be tested;
(4) detecting the detectable label of the nucleic acid probe binding to the nucleic acid molecule to be tested, and determining the base information of the first moiety;
(5) cleaving the ligation between the first moiety of the nucleic acid probe and the linker, and eluting the rest of the nucleic acid probe except the first moiety;
preferably, further comprising the following steps:
(6) repeating the above steps (2) to (4) or (2) to (5).

### Claim 7 (depends on 3)

The kit according to claim 3, wherein the endonuclease is endonuclease IV or endonuclease V.

### Claim 8 (depends on 3)

The kit according to claim 3, wherein the organic phosphide is THPP or TCEP.

### Claim 9 (depends on 1)

The nucleic acid probe combination according to claim 1, wherein the first moiety is located at the 5′-terminal or the 3′-terminal.

### Claim 10 (depends on 1)

The nucleic acid probe combination according to claim 1, wherein the bases of the second moiety are 3 to 15 bases, preferably 5 to 12 bases, and more preferably 5 to 10 bases, particularly preferably 6 to 9 bases.

### Claim 11 (depends on 1)

The nucleic acid probe combination according to claim 1, wherein the detectable label is a fluorophore; preferably one or more selected from the group consisting of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF688;
preferably, the detectable label is ligated to the second moiety;
preferably, the detectable label is ligated to 3′-OH at the end of the second moiety;
preferably, the detectable label is ligated to 3′-OH at the end of the second moiety via a phosphoester bond.

### Claim 12 (depends on 1)

The nucleic acid probe combination according to claim 1, wherein the linker is selected from the group represented by the following Formula IV to Formula IX:
wherein, in Formula IV, R1 is selected from a group consisting of H, OH, C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl; R2 is selected from a group consisting of H, OH, F, Cl, and Br.

### Claim 13 (depends on 1)

The nucleic acid probe combination according to claim 1, wherein the molar ratio of the first group of nucleic acid probes:the second group of nucleic acid probes:the third group of nucleic acid probes:the fourth group of nucleic acid probes is 1:4:4:1.