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Published on: October 25, 2016
Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain
E Y Loh1, J F Elliott, S Cwirla
1Departments of Medicine and Microbiology and Immunology, Stanford University School of Medicine, CA 94305-5402.
Abstract:
In the polymerase chain reaction (PCR), two specific oligonucleotide primers are used to amplify the sequences between them. However, this technique is not suitable for amplifying genes that encode molecules where the 5' portion of the sequences of interest is not known, such as the T cell receptor (TCR) or immunoglobulins. Because of this limitation, a novel technique, anchored polymerase chain reaction (A-PCR), was devised that requires sequence specificity only on the 3' end of the target fragment. It was used to analyze TCR delta chain mRNA's from human peripheral blood gamma delta T cells. Most of these cells had a V delta gene segment not previously described (V delta 3), and the delta chain junctional sequences formed a discrete subpopulation compared with those previously reported.
Insights
Anchored PCR (A-PCR) enables gene amplification without knowing the 5' sequence, unlike traditional PCR. This method identified a novel V delta 3 gene segment in human T cell receptor delta chain mRNA from peripheral blood gamma delta T cells.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Standard polymerase chain reaction (PCR) requires known sequences at both ends of a target for amplification.
- This limitation hinders the analysis of genes encoding molecules like T cell receptors (TCRs) or immunoglobulins, where the 5' sequence is often unknown.
- A novel method, anchored PCR (A-PCR), was developed to overcome this limitation.
Purpose of the Study:
- To introduce and demonstrate the utility of anchored polymerase chain reaction (A-PCR).
- To analyze T cell receptor (TCR) delta chain messenger RNA (mRNA) from human peripheral blood gamma delta T cells.
- To identify novel gene segments and characterize junctional sequences within the TCR delta chain.
Main Methods:
- Development of anchored polymerase chain reaction (A-PCR), which requires sequence specificity only at the 3' end of the target fragment.
- Application of A-PCR to analyze TCR delta chain mRNA from human peripheral blood gamma delta T cells.
- Sequencing and analysis of amplified TCR delta chain fragments to identify gene segments and junctional sequences.
Main Results:
- A previously undescribed V delta gene segment, designated V delta 3, was identified in most analyzed gamma delta T cells.
- The delta chain junctional sequences from these cells formed a distinct subpopulation.
- A-PCR proved effective for amplifying and analyzing TCR delta chain mRNA when 5' sequences are unknown.
Conclusions:
- Anchored PCR (A-PCR) is a valuable tool for amplifying unknown sequences, particularly in the context of immune system gene analysis.
- The identification of V delta 3 suggests greater diversity within the T cell receptor repertoire than previously recognized.
- Junctional sequence analysis in gamma delta T cells reveals specific patterns and subpopulations.
