Sequencing method

US9771615B2 · kind B2 · utility

Assignee

• BASE4 INNOVATION LTD · GB

Inventors

• Cameron Alexander FRAYLING · St Ives, GB
• Barnaby Balmforth · Cambridge, GB
• Bruno Flavio Nogueira de Sousa Soares · Cambridge, GB
• Thomas Henry ISAAC · St Ives, GB
• Boris Breiner · St Ives, GB
• Alessandra Natale · Cambridge, GB
• Michele Amasio · Cambridge, GB

Key dates

• Filing date: Oct 4, 2013
• Grant date: Sep 26, 2017
• Expiry date: May 21, 2034

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/6869
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Disclosed is a method for sequencing a polynucleotide analyte comprising: •a. generating a stream of droplets containing a single nucleotide wherein the order of single nucleotides in the droplet stream corresponds to the sequence of nucleotides in the analyte; •b. introducing into each droplet a plurality of biological probe types each type comprising a different label in an undetectable state and being adapted to capture a different single nucleotide; •c. causing the single nucleotide contained in the droplet to bind to its complementary probe and •d. causing the label to be released from the probe that has bound the nucleotide in a detectable state. The probe is a dumbbell shaped probe comprising fluorescent donor and quencher labels and a single nucleotide gap. After gap repair by a polymerase and a ligase, a restriction enzyme recognition site is cleaved by a restriction enzyme, followed by exonuclease digestion to release the labels.