Methods of Generating Libraries of Nucleic Acid Sequences for Detection via Fluorescent in Situ Sequ

• US 20190330617A1
• Filed: 07/01/2019
• Published: 10/31/2019
• Est. Priority Date: 08/31/2016
• Status: Active Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Daugharthy, Evan R., Terry, Richard C., Pruitt, Benjamin W., Turczyk, Brian M.
• Application Number:
  US16/458,268
• Publication Number:
  US 20190330617A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/327 RNAse, e.g. RNAseH
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

The present disclosure provides a number of targeted nucleic acid FISSEQ library construction methods. Targeted FISSEQ can exhibit several benefits, such as enhanced sensitivity and/or shorter assay time in the detection, identification, quantification, and/or determining the nucleotide sequence of the target species, relative to “random” or “whole-omic” detection via FISSEQ.

Claims

1-102. -102. (canceled)

103. A method for in situ nucleic acid sequence detection or identification of one or more target nucleic acid molecules of a cell, comprising:

(a) providing said cell comprising a reaction mixture comprising said one or more target nucleic acid molecules and a plurality of probes, wherein said plurality of probes comprises a plurality of target-specific sequences, a plurality of adaptor sequences and a plurality of barcode sequences, wherein a probe of said plurality of probes comprises;

(i) a target-specific sequence of said plurality of target-specific sequences that is complementary to a target sequence of a target nucleic acid molecule of said one or more target nucleic acid molecules;

(ii) an adaptor sequence of said plurality of adaptor sequences coupled to said target-specific sequence, wherein said adaptor sequence is for conducting an amplification reaction on said probe when said target-specific sequence is hybridized to said target sequence; and

(iii) a barcode sequence of said plurality of barcode sequences coupled to said adaptor sequence, wherein said barcode sequence is configured to allow detection or identification of said target sequence or at least a portion of said target nucleic acid molecule, and wherein said plurality of barcode sequences are different across said plurality of probes;

(b) within said cell, subjecting said reaction mixture to conditions sufficient to permit said target-specific sequence to hybridize to said target sequence; and

(c) using said barcode sequence to detect or identify said target sequence or said at least said portion of said target nucleic acid molecule.

104. The method of claim 103, wherein said cell is fixed.

105. The method of claim 103, wherein said cell is integrated with a hydrogel.

106. The method of claim 103, wherein said target nucleic acid molecule is a ribonucleic acid molecule, and wherein (b) further comprises subjecting said sequence to conditions sufficient to perform reverse transcription on said sequence to yield a complementary deoxyribonucleic acid molecule.

107. The method of claim 103, wherein said plurality of barcode sequences permits identification of different target sequences of different target nucleic acid molecules of said one or more target nucleic acid molecules.

108. The method of claim 103, wherein said plurality of adaptor sequences comprise identical sequences across said plurality of probes.

109. The method of claim 103, wherein said adaptor sequence is complementary to a primer for conducting said amplification reaction.

110. The method of claim 109, further comprising, prior to (c), binding said primer to said adaptor sequence.

111. The method of claim 110, further comprising, prior to (c), conducting said amplification reaction on said probe when said target-specific sequence is hybridized to said target sequence.

112. The method of claim 103, wherein said probe is a circular probe.

113. The method of claim 103, wherein said target-specific sequence, said adaptor sequence, and said barcode sequence are arranged contiguously from 3′

end to 5′

end of said probe.

114. The method of claim 103, further comprising circularizing said probe.

115. The method of claim 114, wherein said circularizing comprises ligating a 3′

end of said probe to a 5′

end of said probe when said target-specific sequence of said probe is hybridized to said target sequence.

116. The method of claim 115, wherein said circularizing comprises ligating said 3′

end to said 5′

end using a ligase and a splint oligonucleotide independent of said target nucleic acid molecule.

117. The method of claim 115, wherein said circularizing comprises extending said 3′

end of said probe with aid of a reverse transcriptase or a polymerase to yield an extended product, and ligating ends of said extended product together.

118. The method of claim 103, wherein said reaction mixture further comprises a plurality of nucleic acid-binding proteins, which plurality of nucleic acid-binding proteins mediate binding of said plurality of probes onto said one or more target nucleic acid molecules.

119. The method of claim 103, wherein said reaction mixture further comprises a hybridization reaction enhancing agent that enhances a rate of a hybridization reaction between said target nucleic acid molecule and said probe.

120. The method of claim 119, further comprising, subsequent to (b), removing said hybridization reaction enhancing agent from said cell.

121. A method for enhancing a hybridization reaction in a cell or cellular matrix, comprising:

(a) providing said cell or cellular matrix and a reaction mixture, comprising (i) a target nucleic acid molecule, (ii) a probe having sequence complementarity with a target sequence of said target nucleic acid molecule, and (iii) a hybridization reaction enhancing agent comprising a polymer backbone, wherein said hybridization reaction enhancing agent enhances a rate of a hybridization reaction between said target nucleic acid molecule and said probe having sequence complementarity with said target sequence of said target molecule;

(b) subjecting said reaction mixture to conditions sufficient to conduct said hybridization reaction between said target nucleic acid molecule and said probe having sequence complementarity with said target sequence of said target nucleic acid molecule, wherein during said hybridization reaction, said hybridization reaction enhancing agent facilitates said hybridization reaction between said target nucleic acid molecule and said probe having sequence complementarity with said target sequence of said target molecule; and

(c) removing said hybridization reaction enhancing agent from said cell or cellular matrix.

122. The method of claim 121, wherein said removing in (c) comprises washing said hybridization reaction enhancing agent away from said cell or cellular matrix.