Compositions and methods for analyte detection

Patent metadata

Field	Value
Publication number	US-2023212657-A1
Application number	US-202217875211-A
Country	US
Kind code	A1
Filing date	Jul 27, 2022
Priority date	Jan 29, 2020
Publication date	Jul 6, 2023
Grant date	—

Abstract

The present disclosure provides methods, systems and compositions for detecting nucleic acid sequences in a biological sample having a three-dimensional matrix. The present disclosure also provides methods, systems and compositions for processing a biological sample for use in nucleic acid sequence detection

First claim

1 .- 149 . (canceled)

150 . A method for processing a biological sample, comprising:
(a) providing said biological sample comprising a polypeptide analyte within a synthetic three-dimensional (3D) matrix, which synthetic 3D matrix comprises an attachment moiety; (b) contacting said biological sample with a tethering molecule, wherein said tethering molecule comprises (i) an antibody or fragment thereof conjugated to a nucleic acid barcode and (ii) a tethering moiety; and (c) binding said polypeptide analyte with said tethering molecule; thereby attaching said analyte to said synthetic 3D matrix via said attachment moiety.

151 . The method of claim 150 , wherein said synthetic 3D matrix comprises an additional attachment moiety.

152 . The method of claim 150 , wherein (b) is performed simultaneously with forming of the synthetic 3D matrix.

153 . The method of claim 150 , wherein (b) is performed after forming of the synthetic 3D matrix.

154 . The method of claim 150 , wherein, in (a), said biological sample comprises an additional analyte within said synthetic 3D matrix.

155 . The method of claim 151 , wherein said additional analyte is a nucleic acid molecule.

156 . The method of claim 155 , further comprising providing an additional tethering molecule, wherein said additional tethering molecule comprises a primer sequence complementary to a sequence of said nucleic acid molecule.

157 . The method of claim 156 , further comprising, hybridizing said primer sequence to said sequence of said nucleic acid molecule.

158 . The method of claim 157 , further comprising, extending said primer sequence hybridized to said sequence of said nucleic acid molecule to generate an extension product.

159 . The method of claim 158 , wherein said extension product is attached to said synthetic 3D matrix.

160 . The method of claim 158 , further comprising hybridizing a probe to said extension product.

161 . The method of claim 161 , wherein said probe is a padlock probe or a molecular inversion probe.

162 . The method of claim 160 , further comprising circularizing said probe.

163 . The method of claim 162 , wherein said circularizing comprises ligating a 3′ end and a 5′ end of said probe.

164 . The method of claim 160 , further comprising subjecting said probe to an amplification reaction to generate an amplification product.

165 . The method of claim 164 , wherein said amplification reaction is rolling circle amplification.

166 . The method of claim 164 , wherein said amplification reaction incorporates a modified base to said amplification product.

167 . The method of claim 166 , wherein said modified base is 5-azidomethyl-dUTP.

168 . The method of claim 151 , wherein said attachment moiety and said additional attachment moiety are different.

169 . The method of claim 156 , wherein said additional tethering molecule (i) flows through said synthetic 3D matrix and comes in contact with said additional analyte, and (ii) attaches to said additional analyte and said additional attachment moiety, thereby attaching said additional analyte to said synthetic 3D matrix.

Assignees

• 10X Genomics Inc

Inventors

• Daugharthy Evan
• Terry Richard
• Camplisson Conor
• Barclay David
• Miselis Tyler
• Chen Hong
• Droz Shoshoni

Classifications

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title