Historically, T Cell Receptor Gamma Chain Gene Rearrangement Assays have been used to:

1. Identify clonality highly suggestive of T-cell and some immature B-cell malignancies
2. Determine lineage involvement in mature lymphoproliferative disorders
3. Monitor and evaluate disease recurrence

Invivocribe Technologies' assay are standardized PCR-based tests. Each test comes with a Standard Operating Procedure (SOP), an interpretation guide, master mixes, and controls. Master mixes are composed of a buffered magnesium solution, deoxynucleotides, and multiple primers that target the gene segments of interest. Multiple primers are used to ensure a more comprehensive testing approach necessary to reliably identify clonal rearrangements. These tests are complete with the exception of Taq DNA Polymerase, which is not provided. A single thermocycler program and similar detection methods are used within each series of test to improve consistency, reduce human error, and facilitate cross training.

Standard Protocol.
55ul Total PCR Reaction Volume:

1. Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.
2. In a containment hood or dead air box remove an appropriate aliquot to clean, sterile microfuge tube (one tube for each of the master mixes). Aliquot volumes should be 50ul for each sample + 150ul for the positive, negative and no template controls. We recommend adding an additional 20ul to correct for pipetting errors.
3. Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase (0.25ul of either AmpliTaq Gold or AmpliTaq @ 5U/ul per 55ul total PCR reaction volume) to each of the master mixes and gently mix by inverting several times or gently vortexing.
4. Aliquot 50ul of master mix to individual wells of a PCR plate.
5. Add 5ul of DNA from the unknown and control samples to individual tubes or wells containing the respective master mix reactions, and pipette up and down several times to mix. Amplify target DNA using the universal thermocycler program.

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The T cell receptor gamma (TCRG) chain locus spans 160 kilobases on chromosome 7 (7p14). The locus consists of 14 variable (Vg) gene segments in 6 subgroups, and 5 joining (Jg) gene segments interspersed between 2 constant (Cg) gene segments. However, the repertoire of functional TCRG molecules is limited to 4-6 functional Vg gene segments that belong to 2 subgroups. Rearrangement of the variable (Vg) and joining (Jg) gene segments of the TCRG locus results in Vg-Jg products of unique length and sequence. Clonal TCRG rearrangements can be most rapidly identified by analyzing the size distribution of DNA products amplified from conserved sequences that flank this Vg-Jg region. DNA isolated from a normal heterogeneous population of polyclonal T cells produces a Gaussian distribution (bell-shaped size curve) of amplified products. DNA amplified from a clonal T cell population generates one or two product(s) of unique size that reflects proliferation of a single rearranged clone.

Sample genomic DNA is amplified using two master mixes that independently target conserved regions within the variable (Vg) and joining (Jg) regions that flank the unique, hypervariable, antigen-binding, complementarity determining region 3 (CDR3). This assay targets Vg1-9 and Jg gene segments. Positive and negative DNA controls, as well as an internal Amplification Control master mix, are included. The limit of detection of this assay is approximately one clonal T cell in a background of a hundred normal cells. Clonality is indicated if either master mix generates clonal band(s). PCR products of master mixes 1 and 2 are differentially labeled with NED & HEX, respectively, and can be analyzed using any platform that has the ability to detect these fluorochromes such as the Applied Biosystems ABI 310 and ABI 3100. Click to read more

This product tests genomic DNA. DNA can be extracted from the following specimens:

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

This product requires nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). No license under these patents to use amplification processes or enzymes is conveyed expressly or by implication to the purchaser by the purchase of these products. The assays described herein are not approved by any regulatory agency for clinical use. These assays are not for diagnostic or therapeutic use. This product is sold FOR RESEARCH USE ONLY; not for use in diagnostic procedures.