Method of measuring adaptive immunity
First Claim
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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.
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Abstract
A method of measuring immunocompetence is described. This method provides a means for assessing the effects of diseases or conditions that compromise the immune system and of therapies aimed to reconstitute it. This method is based on quantifying T-cell diversity by calculating the number of diverse T-cell receptor (TCR) beta chain variable regions from blood cells.
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Citations
9 Claims
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1. A method for generating a clonotype profile of rearranged T cell receptor (TCR) and/or immunoglobulins (Ig) in a biological sample comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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Specification