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Methods for combining single cell gene expression mapping and targeted RNA or c-DNA sequencing using padlock oligonucleotides

An oligonucleotide and gene expression technology, applied in the field of sequencing and positioning RNA or c-DNA strands, can solve the problem of loss of original position

Pending Publication Date: 2022-01-14
ミルテニイビオテックベーファーウントコーカーゲー
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  • Summary
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the original position at the tissue or single-cell level is also often lost
Methods to capture both sequence and spatial information at near-single-cell resolution remain formidable challenges

Method used

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  • Methods for combining single cell gene expression mapping and targeted RNA or c-DNA sequencing using padlock oligonucleotides
  • Methods for combining single cell gene expression mapping and targeted RNA or c-DNA sequencing using padlock oligonucleotides
  • Methods for combining single cell gene expression mapping and targeted RNA or c-DNA sequencing using padlock oligonucleotides

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Embodiment Construction

[0031] This method combines the use of oligonucleotides that form padlocks upon hybridization (padlock probes) and their sequential hybridization for detection of bound probes with the sequencing of targeted portions of RNA or DNA transcripts at the cellular level , with fewer restrictions in terms of the amount of transcripts that can be analyzed and the length of sequences.

[0032] In this approach, gap-filling padlock probes containing one or more barcoded regions in their cores are used both as FISH probes and to capture fractions of RNA that can be sequenced. The padlock probe has proven to be very successful in polymerizing short nucleic acid moieties to which it has hybridized. Most padlock methods begin by reverse transcribing the target into cDNA.

[0033] Hybridization of the padlock probe to the DNA or RNA strand is followed by a gap-filling step in which the reverse polymerase, starting from the hybridized 5' portion of the probe, fills the open portion between the...

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Abstract

Microscopic imaging of multiple mRNAs is allowed to be parsed at the single cell level, providing valuable information about transcript amount and localization, which is a key factor for understanding tissue heterogeneity, molecular generation and disease treatment. The present invention describes a method (Fly FISH) that combines the use of padlock oligonucleotides as fluorescence in situ hybridization (FISH) probes for detection with the sequencing of targeting moieties of RNA or cDNA transcripts at cellular level with less limitations in the amount of transcripts and sequence length that can be analyzed. Padlock probes containing various barcodes in its core are used both as FISH probes, as well as for capturing RNA portions that can be sequenced. The same barcode can be used to selectively elicit rolling circle amplification and amplify a subset of transcripts from a particular region, which has been labeled as a transcript of interest during the detection step.

Description

technical field [0001] The present invention relates to methods for sequencing and localizing RNA or c-DNA strands by selective amplification of padlock oligonucleotides comprising barcode regions. Background technique [0002] The padlock oligonucleotide has proven to be very successful in polymerizing short nucleic acid moieties to which it has hybridized. Most padlock methods begin by reverse transcribing the target into cDNA. [0003] The padlock method is for example disclosed in: Lee et al. "Highly multiplexed subcellular RNAsequencing in situ", Science. 2014 Mar. 21; 343(6177): 1360-1363. doi:10.1126 / science.1250212, or Nils Schneider and "Efficient In Situ Detection of mRNAs using the Chlorella virus DNA ligase for Padlock ProbeLigation" by Matthias Meier; February 5, 2020 - Cold Spring Harbor Laboratory Press. [0004] A Comprehensive Assay for Targeted Multiplex Amplification of Human DNA Sequences is disclosed in PNAS, submitted for review on February 19, 2008 b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2525/307C12Q2531/125C12Q2563/107C12Q2535/122C12Q1/6841C12Q1/6853C12Q2525/179C12Q2543/101C12Q1/6825
Inventor R·皮纳德S·霍索诺
Owner ミルテニイビオテックベーファーウントコーカーゲー