| CPC C40B 40/08 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6869 (2013.01); C40B 50/00 (2013.01); C40B 50/06 (2013.01); C12Q 2600/16 (2013.01)] | 5 Claims |
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1. A composition for multiplex amplification of an immune repertoire in a sample, comprising:
genomic DNA from a biological sample, a DNA polymerase, dNTPs, and 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 3 (FR3) 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 1 (FR1) within the V gene; and
ii) 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 or an antibody receptor; and wherein each set of i) and ii) primers directed to the same target immune receptor is configured to amplify the target immune receptor repertoire, wherein each of the plurality of V gene primers and/or the plurality of J gene primers includes two or more modified 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, and wherein each of the plurality of V gene primers and/or the plurality of J gene primers further has any one or more of the following criteria:
(1) length is about 15 to about 40 bases in length;
(2) Tm of from above 60° C. to about 70° C.;
(3) has low cross-reactivity with non-target sequences present in the sample;
(4) at least the first four nucleotides (going from 3′ to 5′ direction) are non-complementary to any sequence within any other primer present in the same reaction; and
(5) are non-complementary to any consecutive stretch of at least 5 nucleotides within any other produced target amplicon.
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