Array and method for detecting spatial information of nucleic acids

Patent metadata

Field	Value
Publication number	US-12516446-B2
Application number	US-202017611099-A
Country	US
Kind code	B2
Filing date	May 14, 2020
Priority date	May 15, 2019
Publication date	Jan 6, 2026
Grant date	Jan 6, 2026

Abstract

Provided are a method for detecting spatial information of nucleic acids in a sample, as well as a nucleic acid array used in the method and a method for producing the nucleic acid array.

First claim

What is claimed is:

1 . A method for generating a nucleic acid array for detecting spatial information of a nucleic acid in a biological sample, which comprises the following steps:
(1) providing multiple kinds of carrier sequences, each kind of carrier sequence comprises a plurality of copies of the carrier sequence, and the carrier sequence in the direction from 5′ to 3′ comprises a positioning sequence and a first immobilization sequence, the positioning sequence has a unique nucleotide sequence corresponding to the position of the kind of carrier sequence on the array; the first immobilization sequence allows annealing to its complementary nucleotide sequence and initiating an extension reaction; (2) ligating the multiple kinds of carrier sequences to a surface of a solid support; (3) providing a first primer, and perform a primer extension reaction by using the carrier sequence as a template, so that a region of the first immobilization sequence and the positioning sequence of the carrier sequence forms a double strand, wherein the strand that hybridizes to the carrier sequence is a first nucleic acid molecule, the first nucleic acid molecule in the direction from 5′ to 3′ comprises a complementary sequence of the first immobilization sequence and a complementary sequence of the positioning sequence; wherein, the first primer comprises a first immobilization sequence complementary region at its 3′ end, the first immobilization sequence complementary region comprises a complementary sequence of the first immobilization sequence or a fragment thereof, and has a free 3′ end; and wherein in step (1), the multiple kinds of carrier sequences are provided by the following steps: (i) providing multiple kinds of carrier sequence templates, the carrier sequence template comprising a complementary sequence of the carrier sequence; (ii) perform a nucleic acid amplification reaction by using each kind of carrier sequence template as a template, to obtain an amplification product of each kind of carrier sequence template, the amplification product comprising a plurality of copies of the carrier sequence; and wherein said each kind of carrier sequence comprising the same positioning sequence occupies an area having a diameter of less than 1 micrometer.

2 . The method according to claim 1 , wherein
rolling circle amplification is performed to obtain a DNB formed by a concatemer of the carrier sequence; or, bridge PCR amplification, emulsion PCR amplification or multiple strand displacement amplification is performed to obtain a DNA cluster formed by a clone population of the carrier sequence.

3 . The method according to claim 1 , wherein the method further comprises the following steps:
(4) providing a second nucleic acid molecule, the second nucleic acid molecule comprising a capture sequence; the capture sequence is capable of hybridizing with the whole or part of the nucleic acid to be captured, which comprises: (a) an oligonucleotide sequence capable of capturing mRNA; (b) a random or degenerate oligonucleotide sequence; or, (c) a specific sequence for a specific target nucleic acid; and, the capture sequence has a free 3′ end to enable the second nucleic acid molecule to function as an extension primer, (5) ligating the second nucleic acid molecule to the first nucleic acid molecule.

4 . The method according to claim 3 , wherein the second nucleic acid molecule in the direction from 5′ to 3′ comprises an immobilization region and a capture sequence, and the immobilization region comprises a double-stranded DNA sequence.

5 . The method according to claim 3 , wherein each carrier sequence further comprises a second immobilization sequence at its 5′ end, and the second immobilization sequence allows annealing to its complementary nucleotide sequence; the method further comprises the following steps:
(4) providing a second nucleic acid molecule, the second nucleic acid molecule in the direction from 5′ to 3′ comprising a complement of second immobilization sequence and a capture sequence;
the complement of second immobilization sequence allows hybridization to its complementary nucleotide sequence;
the capture sequence is capable of hybridizing with the whole or part of the nucleic acid to be captured, and comprises: (a) an oligonucleotide sequence capable of capturing mRNA; (b) a random or degenerate oligonucleotide sequence; or, (c) a specific sequence for a specific target nucleic acid; and, the capture sequence has a free 3′ end to enable the second nucleic acid molecule to function as an extension primer;
(5) hybridizing the complement of second immobilization sequence with the second immobilization sequence under a condition that allow annealing, thereby ligating the second nucleic acid molecule to the carrier sequence;
(6) optionally, ligating the first nucleic acid molecule and the second nucleic acid molecule that are hybridized to the carrier sequence respectively.

6 . The method according to claim 3 , wherein, in step (3), the first primer further comprises a unique molecular identifier (UMI) at the 5′ end of its first immobilization sequence complementary region, so that the first nucleic acid molecule comprises the UMI sequence at the 5′ end of its complement of first immobilization sequence; or, in step (4), the second nucleic acid molecule further comprises a UMI sequence, and the UMI sequence is located at the 5′ end of the capture sequence;
and wherein the UMI sequence is a nucleotide sequence consisting of at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides N, each N is independently any one of A, C, G and T.

7 . The method according to claim 1 , wherein:
in step (1), the carrier sequence further comprises a capture sequence template located upstream of the positioning sequence, the capture sequence template comprises a complementary sequence of the capture sequence, and the capture sequence can hybridize to the whole or part of the nucleic acid to be captured, which comprises: (a) an oligonucleotide sequence capable of capturing mRNA; (b) a random or degenerate oligonucleotide sequence; or, (c) a specific sequence for a specific target nucleic acid; and the first immobilization sequence of the carrier sequence also comprises a cleavage site, and the cleavage is selected from enzymatic cleavage with nicking enzyme, enzymatic cleavage with USER enzyme, photocleavage, chemical cleavage or CRISPR-based cleavage; in step (3), a region of the first immobilization sequence, the positioning sequence and the capture sequence template of the carrier sequence forms a double strand, so that the first nucleic acid molecule in the direction from 5′ to 3′ comprises a complement of first immobilization sequence, a complement of positioning sequence and a capture sequence; wherein, the first primer in the direction from 5′ to 3′ comprises a binding region, an cleavage region, and a first immobilization sequence complementary region, the binding region comprises a linker capable of ligating to the surface of the solid support, and the cleavage region comprises a cleavage site; and, the method further comprises the following steps: (4) ligating the first primer to the surface of the solid support; wherein, steps (3) and (4) are performed in any order; (5) optionally, performing cleavage at the cleavage site contained in the first immobilization sequence of the carrier sequence to digest the carrier sequence, so that the extension product in step (3) is separated from the template where such extension product is formed, and the first nucleic acid molecule is therefore ligated to the surface of the solid support.

8 . The method according to claim 7 , wherein, in step (1), the cleavage site contained in the f

Assignees

• Bgi Shenzhen
• Mgi Tech Co Ltd

Inventors

• Chen Ao
• Xu Xun
• Liao Sha
• Yang Jin
• Liu Longqi
• Wang Ou
• Li Yuxiang
• Tang Guoxin
• Jiang Yuan
• Xu Chongjun
• Ni Ming
• Zhang Wenwei
• Drmanac Radoje
• Drmanac Snezana

Classifications

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12Q2533/101

  Primer extension · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6841

  In situ hybridisation · CPC title

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title