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TCR beta PCR from crude preparations for restriction digest or sequencing
1Vermont Cancer Center Genetics Laboratory, University of Vermont, Burlington, 05401, USA.
Environmental and Molecular Mutagenesis
|January 1, 1996
Summary
We developed a novel method to determine T cell clonality using PCR-amplified T cell receptor beta gene. This technique simplifies identifying T cell receptor beta chain sequences and clonality from small cell samples.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- T cell specificity relies on variable (V), diversity (D), and junctional (J) gene segments.
- T cell clones (clonality) can arise in blood or tissue following T cell proliferation.
- Accurate clonality determination is crucial for distinguishing mutants in mutation assays.
Purpose of the Study:
- To introduce a novel, rapid method for determining T cell clonality.
- To utilize restriction digests of PCR-amplified T cell receptor beta gene cDNA.
- To enable easy identification of Vbeta and CDR3 sequences from limited cell numbers.
Main Methods:
- PCR amplification of the T cell receptor beta gene using a consensus primer from cell pellets or RNA.
- Restriction digestion of the PCR-amplified T cell receptor beta cDNA.
- Direct sequencing of the PCR product for Vbeta and CDR3 identification.
Main Results:
- Successfully demonstrated the method's utility on eight T cell clones from two individuals.
- Identified a clone of three identical isolates (3-mer) and a clone of two identical isolates (2-mer) via restriction digests.
- Confirmed clonality using two distinct restriction enzymes.
Conclusions:
- The developed method offers a simpler and faster approach to T cell clonality determination compared to traditional techniques like Southern blotting.
- This technique facilitates efficient analysis of T cell receptor beta gene rearrangements.
- The method is suitable for analyzing clonality in small T cell populations.