T-Cell Receptor Gamma Gene Rearrangement Assay - ABI Fluorescence Detection

Product Details

• Summary of Explanation of the Test

  SUMMARY:

  Invivoscribe’s Gene Rearrangement Assays represent a new approach to PCR-based testing. These assays utilize a series of standardized master mixes that have been carefully optimized. Several master mixes are used to test for rearrangements at each receptor locus, and each master mix targets a different conserved region within the receptor gene. This comprehensive testing approach maximizes identification of clonal rearrangements. A single universal thermocycler program and similar detection methodologies are used with all of our tests; this improves consistency and facilitates cross-training. For example, the three master mixes included in the immunoglobulin heavy chain (IGH) gene rearrangement assays target the joining region and all three of the conserved framework regions within the IGH gene.  Two master mixes in the T-cell gene rearrangement assays target multiple V and J exon regions within the T-cell receptor gamma chain locus. These robust assays can be used to test DNA extracted from virtually any source, and amplification controls, included in the testing kits, ensure that the quality and quantity of input DNA is adequate to yield a valid result.

  BACKGROUND:

  Polymerase chain reaction (PCR) assays are routinely used for the identification of clonal B-cell and T-cell populations. These tests amplify the DNA between primers that target the conserved framework (FR) and joining (J) regions. These conserved regions lie on either side of an area within the V-J region where programmed genetic rearrangements occur during maturation of all B and T lymphocytes. The antigen receptor genes that undergo rearrangement are the immunoglobulin heavy chain & light chains genes in B-cells, and the T cell receptor genes in T-cells. Each B- and T-cell has a single productive V-J rearrangement that is unique in both length and sequence. Therefore, when this region is amplified using DNA primers that flank this region, a clonal population of cells yields one or two prominent amplified products (amplicons) within the expected size range. Two products are produced in cases when the initial rearrangement was non-productive and was followed by rearrangement of the other homologous chromosome. In contrast, DNA from a normal or polyclonal (many clones) population produces a bell-shaped curve of amplicon products (or Gaussian distribution) that reflect the heterogeneous population of V-J region rearrangements.

  Since the antigen receptor genes are polymorphic (consisting of a heterogeneous population of related DNA sequences), it is difficult to employ a single set of DNA primer sequences to target all of the conserved flanking regions around the V-J rearrangement. N-region diversity and somatic mutation further scramble the DNA sequences in these regions. Therefore, multiple DNA primers, which target several FR regions, are required to identify the majority of clonal rearrangements. As indicated, clonal rearrangements are identified as prominent, single-sized products within the smear of different-sized amplicon products that form a Gaussian distribution around a statistically-favored, average-sized rearrangement. As expected, primers that amplify from the different FR regions, which are located sequentially along the heavy chain gene, produce a correspondingly different size-range of V-J products.

  Gel electrophoresis is commonly used to resolve the different-sized amplicon products and ethidium bromide or other DNA intercalating dyes to stain and detect these products. A powerful alternative method is use of differential fluorescence detection with primers conjugated with fluorescent dyes that correspond to different targeted regions. Reaction products from several different master mixes can be pooled, fractionated using capillary electrophoresis, and detected simultaneously. This detection system results in unsurpassed sensitivity, single base resolution, differential product detection and quantification. In addition, the laboratory can eliminate the use of agarose and polyacrylamide gels, as well as the use of carcinogens such as ethidium bromide. Further, differential detection allows accurate, reproducible and objective interpretation of primer-specific products and automatic archiving of data.  The limit of detection of this assay has been determined to be better than 1 clonal cell in 100 hundred normal cells, and inter-assay and intra-assay reproducibility in size determination is approximately 0.1 basepairs. This reproducibility and sensitivity allows monitoring and tracking of individual tumors during research or methods development. The automatic archiving of specimen data allows comparison of data collected at different times.

• Specimen Requirements

  This assay tests genomic DNA

  1. 5cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA. Ship at ambient temperature; OR
  2. Minimum 5mm cube of tissue shipped frozen; or at room temperature or on ice in RPMI 1640; OR
  3. 2µg of genomic DNA; OR
  4. Formalin-fixed paraffin embedded tissue or slides.