RNA templated ligation

US11597965B2 · kind B2 · utility

Assignee

• 10x Genomics, Inc. · US

Inventors

• Mats Nilsson · Uppsala, SE
• Malte KÜHNEMUND · Stockholm, SE
• Tomasz KRZYWKOWSKI · Geber, SE

Key dates

• Filing date: Oct 5, 2018
• Grant date: Mar 7, 2023
• Expiry date: Oct 5, 2038

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N2310/532
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.