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A Method for Studying Combinatorial Diversity of v(d)j

A diverse, methodological technique applied in the field of immunology

Active Publication Date: 2016-02-03
ADAPTIVE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0037] However, none of the above approaches can obtain a resolution of V(D)J rearrangements in the immune repertoire, or even of all rearrangements that occur at a particular locus, in a reasonable length of time

Method used

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  • A Method for Studying Combinatorial Diversity of v(d)j
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  • A Method for Studying Combinatorial Diversity of v(d)j

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0177] Embodiment 1: The selection of the position that can be used for the primer of the present invention

[0178] Oligonucleotide Selection Criteria

[0179] The primers that can be used to implement the method of the present invention are selected according to the following conditions: 1) their thermodynamic properties (determined based on algorithms routinely used by those skilled in the art to identify the ability of an oligonucleotide to bind to its target sequence, especially according to number of hydrogen bonds); 2) their thermodynamic compatibility with other primers used in the same tube and in terms of mutual non-hybridization of different primers; 3) their respective positions such that amplicons can be obtained that can be separated size.

[0180] The term "separation" should be understood to mean that the amplicons can be observed individually after being separated according to their size by electrophoresis or any other method. When certain amplicons are to...

Embodiment 2

[0229] Example 2: The scheme using the ImmuTraCkeR kit (Taking the TRB locus as an example)

[0230] The necessary sample volume is optimized according to the nature of the sample (cells, PBMCs, thymus extracts, etc.). For cell samples, 10 6 cells.

[0231] The sequence diagram of the different steps of the scheme is shown in Figure 19 middle.

[0232] 2-A. DNA extraction

[0233] Extraction of high-purity DNA is essential for detection of V-J rearrangements using the ImmunTraCkeR kit. Those skilled in the art know which methods or kits are suitable for this. In particular, those skilled in the art know that the extraction should be performed in the absence of any EDTA or other substances that can inhibit PCR. The inventor proposes to use DNA was extracted using the HighPure PCRPreperationTemplate kit.

[0234] The recommended DNA concentration is 100ng / μl.

[0235] 2-B. Controlling DNA Quality and Determining DNA Quantity

[0236] The absorbance of the sample at...

Embodiment 3

[0255] Example 3: Analysis of rearrangements of all functional J genes at the β locus with a given V gene using a 2-plex PCR

[0256] Figure 2a Analysis of mean GC content in human TRBJ and TRAJ regions is shown. The J2 region has a very high GC content of 60% compared to the average of 40-45%.

[0257] Figure 2b Specific rearrangements of J genes (2 different clusters) at the TRB locus are shown.

[0258] Figure 2c The rationale for selecting primers is described where 1 antisense primer is close to and the other antisense primer is far from the J cluster so that the entire J1 cluster can be located above the J2 cluster.

[0259] Figure 2d is an example of the results obtained using this method.

[0260] The procedure halved the biological material needed and also reduced the cost of the experiment. The time to conduct experiments is also reduced. Furthermore, this allows for simplified reading of gel (or other type of separation) lanes, since the J genes are arra...

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Abstract

Method for in vitro analysis of the diversity of T and / or B lymphocyte repertoire of individual, using genomic DNA from biological sample of individual, comprises amplification of genomic DNA fragments by multiplex polymerase chain amplification reaction, detection of amplification products obtained in amplification step; and interpretation of results. Method for in vitro analysis of the diversity of T and / or B lymphocyte repertoire of an individual, by using genomic DNA obtained from a biological sample of the individual, comprises: amplification of genomic DNA fragments by multiplex polymerase chain amplification reaction of which one multi-n-plex PCR (where n is >= 2) is carried out with a combination of at least 3 primers, comprising at least 2 pairs of primers having the following characteristics (each pair of primers is constituted of a primer specifically hybridizing upstream and / or in a given V or D gene and a primer hybridizing specifically in downstream and / or in a given J gene, to allow the amplification of two fragment characteristics of two different rearrangements of V(D)J or D-J, the primers are thermodynamically compatible, and the primers are selected so that the fragments amplified with the first pair of primers are distinguished from amplified fragments with the second pair of primers); detection of amplification products obtained in amplification step; and interpretation of results. Independent claims are included for: (1) a method of analysis of the diversity of combinatorial rearrangements of the T cell receptor beta (TRB) locus in an individual, comprising combining the analysis of rearrangements V(D)J of the locus by the method and the analysis of incomplete rearrangements of the locus by the method; (2) a method for determining in vitro the level of immune deficiency of an individual, comprising numbering lymphocyte, from a biological sample of the individual; determining the level of combinatorial diversity of the lymphocyte repertoire of individual by implementing the method, from the same sample or another sample from the same individual at the same time; and combining the obtained data; (3) a method for monitoring the evolution of the diversity of the T and / or B lymphocyte repertoire of the individual, comprising measuring the diversity of the individual lymphocyte repertoire, by implementing the method by using two samples of individuals collected at two different dates, and comparing the two samples by evaluating the number S of rearrangements observed in both samples, the number A of rearrangements observed in the latest sample but not the oldest, the number D of rearrangements observed in the oldest sample but not the latest, and the number Z of rearrangements that are observed in any sample; and (4) a kit for the implementation of the method comprising a combination of primers and reagents to perform PCR.

Description

technical field [0001] The present invention relates to the field of immunology. More specifically, the present invention relates to methods and uses thereof for the in vitro analysis of the diversity of T and / or B lymphocyte repertoires in an individual, in particular during treatment follow-up or during the diagnosis and / or prognosis of certain pathological conditions. Background technique [0002] Lymphocytes must have an antigen-specific recognition system in order to function. This factor is important: it determines the specific function of T or B cells. During the early stages of T lymphocyte differentiation, rearrangements of sites encoding the TCR receptor clonotype chains allow the expression of functional receptors. Similarly, in B lymphocytes, the sites encoding the immunoglobulin (Ig) chains are rearranged allowing the expression of functional Ig. [0003] The V(D)J rearrangement mechanism is specific for T and B lymphocytes. The V, D, and J genes encoding TC...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/106C12Q2600/156C12Q2600/16
Inventor 尼古拉斯·贝吕耶塞巴斯蒂安·韦斯布赫
Owner ADAPTIVE BIOTECH
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