Nested PCR Primer Set for Mouse V Gene Amplification
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Solution Overview
Problem
Current PCR technologies face challenges in achieving broad and highly specific amplification of mouse antibody genes due to intra- and inter-family sequence variability, and somatic hypermutation events that affect primer binding sites, leading to inefficient amplification of rearranged V genes from single or small numbers of B cells.
Innovation Solution
A novel set of nested PCR oligonucleotide primers designed to amplify all possible mouse rearranged V H, V K, and V λ genes with minimum cross-reactivity, low degeneracy, high specificity, and sensitivity, using genomic DNA or total RNA from small numbers of B cells, covering all V gene families with minimal mismatches and allowing isotype-independent amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If highly degenerate primers are used to amplify all V gene segments, then the coverage of V gene families is improved, but the specificity decreases leading to cross-family amplification
Solution Approach 1:
The primer set is divided into multiple specific primers, each targeting a particular V gene family (VH1-VH16, VK1-VK19, VL1-VL3), replacing the single highly degenerate primer approach. This segmentation allows each primer to maintain high specificity for its target family while collectively covering all V gene families through the combined primer set.
2Ease of operation
If standard PCR is used with existing primer sets, then the amplification process is simple, but the sensitivity is insufficient to amplify rearranged V genes from single or small numbers of B cells
Solution Approach 1:
The patent implements a nested PCR strategy where a first PCR amplifies the target DNA using outer primers, and a second PCR uses inner primers to amplify a portion of the first PCR product. This nested approach significantly enhances sensitivity, enabling detection from single B cells while maintaining operational simplicity through standardized PCR protocols.
3Reliability
If primers are designed for standard PCR with minimum degeneracy, then the cross-family amplification is reduced, but the sensitivity and detection limit are insufficient for single B cell analysis
Solution Approach 1:
The nested PCR design with specific primers for each V gene family achieves both high specificity and enhanced detection sensitivity. The two-round amplification process allows the first PCR to establish sufficient template quantity while the second PCR provides specific amplification with lower detection limits, enabling single B cell analysis.
4Adaptability or versatility
If somatic hypermutation events occur at primer binding sites, then the natural diversity of B cell repertoire is maintained, but the amplification efficiency decreases due to primer binding site mismatches
Solution Approach 1:
The primers are designed to bind to highly conserved framework regions (FR1, FR2, FR4) of the V genes that exhibit minimal sequence variability even after somatic hypermutation. This local quality approach ensures that primer binding sites remain stable and efficient while the hypervariable regions (CDRs) maintain their diversity for B cell repertoire representation.
Data Source
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AI summary
The present invention provides oligonucleotides for detection of rearrangement of immunoglobulin genes for identifying clonality of cells, cancer cells, hypermutation in immunoglobulin gene, antibody isotype producing cell and/or assaying B cell repertoire in a sample. The oligonucleotides disclosed in the present invention are very specific to the immunoglobulin genes.