US 9,809,813 B2
Method of measuring adaptive immunity
Harlan S. Robins, Seattle, WA (US); Edus H. Warren, III, Bainbridge Island, WA (US); and Christopher Scott Carlson, Kirkland, WA (US)
Assigned to Fred Hutchinson Cancer Research Center, Seattle, WA (US)
Filed by FRED HUTCHINSON CANCER RESEARCH CENTER, Seattle, WA (US)
Filed on Mar. 4, 2016, as Appl. No. 15/61,827.
Application 15/061,827 is a continuation of application No. 14/243,875, filed on Apr. 2, 2014, abandoned.
Application 14/243,875 is a continuation of application No. 12/794,507, filed on Jun. 4, 2010, abandoned.
Claims priority of provisional application 61/220,344, filed on Jun. 25, 2009.
Prior Publication US 2016/0251721 A1, Sep. 1, 2016
Int. Cl. C12Q 1/68 (2006.01); G06F 19/24 (2011.01); C12N 15/10 (2006.01)
CPC C12N 15/1065 (2013.01) [C12Q 1/6869 (2013.01); C12Q 1/6874 (2013.01); C12Q 1/6881 (2013.01); C12Q 1/6883 (2013.01); G06F 19/24 (2013.01); C12Q 2600/16 (2013.01)] 9 Claims
 
1. A method for generating a clonotype profile of rearranged T cell receptor (TCR) and/or immunoglobulins (Ig) in a biological sample comprising:
(a) combining a plurality of V segment primers and a plurality of J segment primers with DNA from a biological sample comprising T cells and/or B cells wherein the V segment primers for TCR amplification comprise the primers of SEQ ID Nos: 1-45, the J segment primers for TCR amplification comprise the primers of SEQ ID Nos: 46-57, the V segment primers for Ig amplification comprise the primers of SEQ ID Nos:443-451 and the J segment primers for amplification of Ig comprise the primers of SEQ ID Nos: 452-467, wherein the terminal “n” position in SEQ ID NO: 1-45 and SEQ ID Nos: 46-57 comprises a universal primer sequence, and wherein SEQ ID Nos:443-451 and SEQ ID Nos: 452-467 comprise a universal primer sequence;
(b) amplifying rearranged TCR and/or Ig using the plurality of V segment primers and J segment primers in a multiplex polymerase chain reaction (PCR) to produce amplified rearranged TCR and/or Ig DNA molecules;
(c) immobilizing said amplified rearranged TCR and/or Ig DNA molecules on a solid surface and performing solid phase PCR to form template clusters on the solid surface; and
(d) sequencing the TCR and/or Ig DNA molecules in the template clusters to produce sequence reads that encompasses a CDR3 encoding region of the TCR and/or Ig thereby generating a clonotype profile TCRs and/or Igs in the biological sample.