Patent ID: 11970787
Assignee: LIFE TECHNOLOGIES CORPORATION
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method for preparing an immune receptor repertoire library, comprising:
A. generating target immune receptor amplicon molecules comprising performing a single multiplex amplification reaction to amplify expressed target immune receptor nucleic acid template molecules using at least one set of:
i) (a) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 1 (FRI) within the V gene,
(b) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 2 (FR2) within the V gene, or
(c) a plurality of V gene primers directed to a majority of different V genes of at least one immune receptor coding sequence comprising at least a portion of framework region 3 (FR3) within the V gene; and
ii) (a) one or more C gene primers directed to at least a portion of a C gene of the at least one immune receptor coding sequence, or
(b) a plurality of J gene primers directed to at least a portion of a majority of different J genes of the at least one immune receptor coding sequence;
wherein each set of i) and ii) primers is directed to coding sequences of the same target immune receptor gene selected from a T cell receptor gene or an antibody receptor gene and wherein performing the amplification using the at least one set of i) and ii) primers results in amplicon molecules representing a target immune receptor repertoire in a sample, thereby generating immune receptor amplicon molecules;
B. treating the amplicon molecules to form blunt-ended amplicon molecules; and
C. ligating at least one adapter to at least one of the treated amplicon molecules, thereby producing a library of adapter-ligated target immune receptor amplicon molecules comprising the target immune receptor repertoire;
wherein each of the plurality of V gene primers, the plurality of J gene primers and/or the one of more C gene primers includes two or more modified cleavable nucleotides within the primer sequence, at least one of which is included near or at the termini of the primer and at least one of which is included at, or about the center nucleotide position of the primer sequence.