Methods and compositions for synchronizing reactions in situ

Patent metadata

Field	Value
Publication number	US-12360105-B2
Application number	US-202217877673-A
Country	US
Kind code	B2
Filing date	Jul 29, 2022
Priority date	Jul 30, 2021
Publication date	Jul 15, 2025
Grant date	Jul 15, 2025

Abstract

The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address issues associated with the heterogeneity of analyte abundance (e.g., gene expression levels) and variations among reactions at different locations of a sample (e.g., amplification reaction starting earlier at one location than another location). In some aspects, a method disclosed herein provides a tighter distribution of signal spot size and intensity in a sample, as compared to methods that result in a wide and heterogeneous size and intensity distribution of signal spots.

First claim

The invention claimed is:

1. A method for analyzing a biological sample, comprising:
(a) contacting the biological sample with a first reaction mixture, wherein:
the biological sample comprises a circular nucleic acid comprising a hybridization region,
the first reaction mixture comprises a polymerase, wherein the biological sample comprises cells or is a tissue sample,
the circular nucleic acid or the polymerase is prebound to a polynucleotide comprising a sequence complementary to the hybridization region, and
the polymerase activity of the polymerase is inhibited; and
(b) contacting the biological sample with a second reaction mixture to allow the polymerase to extend the polynucleotide hybridized to the hybridization region using the circular nucleic acid as a template,
wherein a rolling circle amplification product of the circular nucleic acid is generated in the biological sample.

2. The method of claim 1 , wherein the first reaction mixture is substantially free of deoxynucleoside triphosphates (dNTPs) and/or nucleoside triphosphates (NTPs).

3. The method of claim 1 , wherein the first reaction mixture comprises a non-catalytic cofactor of the polymerase, wherein the non-catalytic cofactor is selected from the group consisting of: calcium, barium, strontium, iron, cobalt, nickel, tin, zinc, and europium.

4. The method of claim 1 , wherein the first reaction mixture is substantially free of a catalytic cofactor of the polymerase, wherein the catalytic cofactor comprises Mg 2+ , Co 2+ , and/or Mn 2+ .

5. The method of claim 4 , wherein the first reaction mixture comprises a chelating agent, wherein the chelating agent comprises EDTA, EGTA, BAPTA, DTPA, or a combination thereof.

6. The method of claim 1 , wherein the polynucleotide comprises a 3′ protective group, thereby protecting the polynucleotide from 3′→5′ exonuclease degradation by the polymerase while allowing priming by the polymerase.

7. The method of claim 1 , wherein the polynucleotide is a primer, and the primer is prebound to the polymerase in the first reaction mixture prior to contacting the biological sample in step (a).

8. The method of claim 1 , wherein the polynucleotide is a primer, and the primer is prebound to the circular nucleic acid in the biological sample prior to contacting the first reaction mixture in step (a).

9. The method of claim 8 , wherein the hybridization region in the circular nucleic acid is a primer hybridization region that hybridizes to the primer, and the circular nucleic acid further comprises a target hybridization region that hybridizes to a target nucleic acid.

10. The method of claim 1 , wherein the polynucleotide is a target nucleic acid, and the target nucleic acid is prebound to the circular nucleic acid in the biological sample prior to and/or during contacting the first reaction mixture in step (a).

11. The method of claim 1 , wherein the second reaction mixture:
(a) comprises a deoxynucleoside triphosphate (dNTP) and/or a nucleoside triphosphate (NTP);
(b) comprises a catalytic cofactor of the polymerase, wherein the catalytic cofactor is a di-cation; and
(c) is substantially free of the polymerase and/or other polymerases.

12. The method of claim 1 , wherein the polynucleotide hybridized to the hybridization region is extended by the polymerase using the circular nucleic acid as a template, thereby generating the rolling circle amplification product.

13. The method of claim 1 , wherein the method comprises imaging the biological sample to detect the rolling circle amplification product.

14. The method of claim 13 , wherein the imaging comprises detecting a signal associated with a fluorescently labeled probe that directly or indirectly binds to the rolling circle amplification product.

15. The method of claim 1 , wherein a sequence of the rolling circle amplification product is analyzed in situ in the biological sample.

16. The method of claim 1 , wherein:
the circular nucleic acid is formed in the biological sample from a probe or a probe set for a target molecule,
the target molecule is genomic DNA, mitochondrial DNA, mRNA or cDNA, and
the probe or probe set for the target molecule comprises a padlock probe that hybridizes to the genomic DNA, mitochondrial DNA, mRNA or cDNA.

17. The method of claim 1 , wherein the first reaction mixture comprises a non-catalytic metal ion.

18. The method of claim 17 , wherein the non-catalytic metal ion is Ca 2+ or Sr 2+ .

19. The method of claim 1 , wherein the polymerase is selected from the group consisting of Phi29 DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRD1 polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, Klenow fragment, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and a variant or derivative thereof.

20. The method of claim 1 , further comprising, between the contacting in step (a) and step (b), a step of removing molecules of the polymerase and/or the polynucleotide that are not bound to the circular nucleic acid from the biological sample.

Assignees

• 10X Genomics Inc

Inventors

• Hernández Neuta Jorge Iván
• Kühnemund Malte
• Marks Patrick J

Classifications

• C12Q1/6876

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

• C12Q1/6813

  Hybridisation assays · CPC title

• C12Q1/6841

  In situ hybridisation · CPC title

• G01N33/5308 Primary

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• C12Q1/6853 Primary

  using modified primers or templates · CPC title