Multiplex PCR V(D)J Diversity Analysis
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Solution Overview
Problem
Current methods for analyzing the diversity of T and B lymphocyte repertoires are inefficient in resolving V(D)J rearrangements, particularly in a timely and routine manner, due to the high number of multiplex PCRs required, which limits their applicability in immunomonitoring and diagnostics.
Innovation Solution
A method involving multiplex PCR with combinations of primers that are thermodynamically compatible, allowing the simultaneous amplification and differentiation of multiple V-J or D-J rearrangements, using high-performance Taq polymerase capable of amplifying large fragments and robust in GC-rich regions, followed by real-time measurement and size separation of amplicons to assess combinatorial and molecular diversity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional methods for analyzing V(D)J rearrangements are used, then comprehensive diversity assessment is achieved, but the analysis time and complexity increase significantly
Solution Approach 1:
The patent segments the V(D)J locus into multiple defined regions (e.g., V, D, J segments) and uses region-specific primers to amplify each segment separately. This segmentation allows comprehensive diversity assessment through multiple targeted amplifications rather than attempting to analyze the entire locus in a single complex reaction, thereby maintaining measurement precision while managing analysis time through systematic processing of divided regions.
2Measurement precision
If multiple multiplex PCRs are performed to resolve all V(D)J rearrangements, then complete repertoire diversity is captured, but the number of reactions and operational complexity increase
Solution Approach 1:
The patent employs universal primers that can bind to conserved sequences across different V(D)J gene families, allowing a single primer set to amplify multiple different rearrangements simultaneously. This multi-functionality enables complete repertoire diversity to be captured through fewer universal multiplex PCRs rather than requiring separate family-specific reactions for each V, D, or J segment combination.
3Ease of manufacture
If standard Taq polymerase is used for amplification, then the procedure is simple and cost-effective, but amplification of large fragments and GC-rich regions is inefficient
Solution Approach 1:
The patent modifies amplification parameters including using extended elongation times (e.g., 15-30 seconds per kilobase), optimizing annealing temperatures, and adjusting MgCl2 concentrations to enhance the performance of standard Taq polymerase. These parameter changes enable reliable amplification of large fragments and GC-rich regions while maintaining the use of cost-effective standard Taq polymerase rather than requiring expensive specialized enzymes.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables rapid and reliable analysis of a large number of V(D)J rearrangements, providing comprehensive insights into lymphocyte repertoire diversity, aiding in immunomonitoring, treatment evaluation, and diagnosis, particularly in oncology-hematology.
Implementation Method 1
amplification of fragments of said genomic DNA by multiplex PCRs
Implementation Method 2
a primer which specifically hybridizes upstream of and/or in a given V or D gene and of a primer which specifically hybridizes downstream of and/or in a given J gene
Implementation Method 3
separation of said products according to their size
Data Source
AI summary
The invention relates to a method for analyzing the diversity of the catalogue of T and/or B lymphocytes in an individual, based on the amplification, from a sample, of genomic DNA fragments by PCR multi-n-plexes, with n≥2, carried out with a combination of at least 3 primers defining at least 2 primer couples, each of which includes a primer specifically hybridizing upstream and/or in a given V or D gene and a primer specifically hybridizing downstream and/or in a given J gene, in order to obtain the amplification of at least two fragments characteristic of two distinct V-J or D-J rearrangements from each primer couple. The invention also relates to the applications of this method, in particular in the treatment follow-up or in the diagnosis and/or prognosis of certain diseases.