Patent ID: 11891651
Assignee: ELEMENT BIOSCIENCES, INC.
Field: Measurement (Instruments)
Classification: CPC C  G | IPC C  G

Claim 15:
16. A method for pairwise sequencing, comprising:
a) providing a support having a plurality of a first surface primers immobilized thereon, wherein individual immobilized first surface primers in the plurality comprise a first portion (SP1-A) and a second portion (SP1-B), and wherein individual first surface primers comprise a 3′ extendible end and lack a nucleotide having a scissile moiety that can be cleaved to generate an abasic site in the first surface primer;
b) contacting the plurality of the first surface primers with a plurality of single stranded linear nucleic acid library molecules, wherein individual library molecules have a universal sequence (SP1-A′) at the 5′ end that binds to the first portion of the immobilized first surface primers and a universal sequence (SP1-B′) at the 3′ end that binds to the second portion of the immobilized first surface primers, and wherein the contacting is conducted under conditions suitable for hybridizing individual library molecules with the immobilized first surface primers to form circularized library molecules, wherein individual circularized library molecules have a gap or nick between the 5′ and 3′ ends of the circularized library molecule;
c) enzymatically closing the gap or nick, thereby forming covalently closed circular molecules that are hybridized to the plurality of immobilized first surface primers;
d) generating a plurality of immobilized single stranded nucleic acid concatemer template molecules by conducting a rolling circle amplification reaction with a plurality of a strand displacing polymerases, and a plurality of nucleotides which include dATP, dCTP, dGTP, dTTP and nucleotides having a scissile moiety that can be cleaved to generate an abasic site, thereby generating a plurality of immobilized single stranded nucleic acid concatemer template molecules, wherein one or more individual concatemer template molecules within the plurality have at least one nucleotide with a scissile moiety, and wherein individual concatemer template molecules are covalently joined to immobilized first surface primers;
e) sequencing the plurality of immobilized concatemer template molecules, thereby generating a plurality of extended forward sequencing primer strands, wherein individual immobilized concatemer template molecules have two or more extended forward sequencing primer strands hybridized thereon;
f) retaining the plurality of immobilized concatemer template molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands that are hybridized to the retained immobilized concatemer template molecules by conducting a primer extension reaction;
g) removing the retained immobilized concatemer template molecules by generating abasic sites in the immobilized single stranded concatemer template molecules at the nucleotide(s) having the scissile moiety and generating gaps at the abasic sites to generate a plurality of gap-containing single stranded nucleic acid concatemer template molecules while retaining the plurality of forward extension strands and retaining the plurality of immobilized first surface primers; and
h) sequencing the plurality of retained forward extension strands, thereby generating a plurality of extended reverse sequencing primer strands, wherein individual forward extension strands have two or more extended reverse sequencing primer strands hybridized thereon.