Method of measuring adaptive immunity

Patent metadata

Field	Value
Publication number	US-9809813-B2
Application number	US-201615061827-A
Country	US
Kind code	B2
Filing date	Mar 4, 2016
Priority date	Jun 25, 2009
Publication date	Nov 7, 2017
Grant date	Nov 7, 2017

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.

First claim

What is claimed:

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.

2. The method of claim 1 wherein the universal primer sequence is a GA2 universal primer sequence.

3. The method of claim 2 wherein the V segment primers for TCR amplification comprise SEQ ID NOS: 58-102 and the J segment primers for TCR amplification comprise SEQ ID Nos: 103-113.

4. The method of claim 1 wherein the total diversity of rearranged TCR is greater than 1×10 6 .

5. The method of claim 1 wherein sequencing in step (d) is done by reversible dye termination chemistry.

6. The method of claim 5 wherein the sequencing reads 30-54 base pair intervals.

7. The method of claim 1 wherein the sample comprises genomic DNA.

8. The method of claim 1 wherein the sample comprises cDNA.

9. The method of claim 1 wherein the biological sample is a blood sample.

Assignees

• Hutchinson Fred Cancer Res

Inventors

• Robins Harlan S
• Warren Iii Edus H
• Carlson Christopher Scott

Classifications

• G06F19/24

  Physics · mapped topic

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6883

  for diseases caused by alterations of genetic material · CPC title