CAT #: 23090020
BCL2/JH t(14;18) (mcr) Mix 2b - Unlabeled
Master mixes are components of complete assays. 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.
55 µl Total PCR Reaction Volume:
- Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.
- 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 50 µl for each sample + 150 µl for the positive, negative and no template controls. We recommend adding an additional 20 µl to correct for pipetting errors.
- Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase (0.25 µl of either AmpliTaq Gold or AmpliTaq @ 5 U/µl per 55 µl total PCR reaction volume) to each of the master mixes and gently mix by inverting several times or gently vortexing.
- Aliquot 50 µl of master mix to individual wells of a PCR plate.
- Add 5 µl 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.
Historically, BCL2/JH t(14;18) Translocation Assays have been used to:
- Distinguish lymphoma from benign lymphoid hyperplasia
- Distinguish follicular lymphoma from other B-cell lymphomas that may have a similar appearance
- Monitor and evaluate disease recurrence and detect residual disease
- Specimen Requirements
This product tests genomic DNA. DNA can be extracted from the following specimens:
- 5cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA; OR,
- Minimum 5mm cube of tissue shipped frozen, at room temperature, or on ice in RPMI 1640; OR,
- Formalin-fixed paraffin embedded tissue or slides.
The BCL2 t(14;18) (q32;q21) translocation is found in 80-90% of follicular lymphomas and 30% of diffuse large cell lymphomas. The translocation is rarely present in other lymphoproliferative diseases. The t(14;18) brings about juxtaposition of BCL2 with the Ig heavy chain joining segment. This leads to a marked increase in expression of BCL2 driven by the Ig heavy chain gene enhancer. The bcl-2 protein inhibits programmed cell death (apoptosis) leading to cell accumulation. The majority of breakpoints on 18q21-22 occur within the major breakpoint region (Mbr) in the 3′ untranslated region of exon 3 (60-70% of the cases), and the minor cluster region (mcr) located 3′ to BCL2 exon 3 (20-25% of the cases). Some breakpoints occur at distant loci and will not be identified by this particular test. Therefore, a negative result does not completely exclude the presence of a BCL2/IGH gene rearrangement in the sample. In comparison, Southern blot analysis requires 1-2 weeks, is significantly less sensitive, and has more restrictive specimen requirements.
Five master mixes are included in this assay kit. Two master mixes target BCL2 Mbr translocations and two target BCL2 mcr translocations. An Amplification Control master mix is also included to ensure the quality and quantity of sample DNA. Positive and negative controls are also included. This assay can be run either in a standard or nested assay format. Using the standard method, the limit of detection is better than one cell in one hundred normal cells. The nested method has a limit of detection better than one t(14;18) positive cell in a background of ten thousand normal cells. Clonality is indicated if just one of the 2nd round master mixes (mixes ending in b) generates clonal band(s). PCR products can be analyzed using standard gel electrophoresis with ethidium staining.
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This product is for Research Use Only; not for use in diagnostic procedures.
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