ZytoLight^® SPEC CDK4/CEN 12 Dual Color Probe

The ZytoLight SPEC CDK4/CEN 12 Dual Color Probe (PL61) is a direct-labeled FISH probe designed for the qualitative detection of amplifications involving the human CDK4 gene and for detecting specific sequences on chromosome 12 in formalin-fixed, paraffin-embedded (FFPE) specimens. It is intended to be used in combination with the ZytoLight FISH-Tissue Implementation Kit.

Principle:

The fluorescence in situ hybridization (FISH) technique detects and visualizes specific nucleic acid sequences using fluorescently-labeled DNA fragments (FISH probes) that co-denature and hybridize with complementary target DNA strands. After stringent washing to remove unbound fragments, the hybridized probes are visualized with a fluorescence microscope utilizing specific excitation and emission filters.In this method, fluorescently labeled locus-specific (CDK4) and centromeric (CEN 12) DNA probes co-denature with target DNA in pretreated formalin-fixed, paraffin-embedded (FFPE) sections (10 µl of probe per slide, sealed with a coverslip and rubber cement). Following denaturation at 75°C for 10 minutes and overnight hybridization at 37°C, stringent washing and DAPI counterstaining enable visualization with color-specific filters according to the ZytoLight FISH-Tissue Implementation Kit protocol.

Components:

ZytoLight^® SPEC CDK4/CEN 12 Dual Color Probe comprises:

• ZyGreen labeled polynucleotides (excitation 503 nm/emission 528 nm) targeting the CDK4 gene region on chromosome 12 (12q13.3).
• ZyOrange labeled polynucleotides (excitation 547 nm/emission 572 nm) targeting the alpha satellite centromeric region D12Z3 of chromosome 12.
• Formamide-based hybridization buffer.

The probe set enables qualitative detection of the CDK4 gene region and chromosome 12 centromere in formalin-fixed, paraffin-embedded (FFPE) tissue specimens by FISH. Used with ZytoLight^® FISH-Tissue Implementation Kit for specimen pretreatment (dewaxing, proteolysis), denaturation/hybridization, and post-hybridization processing (washing, counterstaining).

In a normal interphase nucleus, two green signals (for CDK4) and two orange signals (for CEN 12) are expected. Amplifications of the CDK4 gene region will result in an increased number of green signals or clusters. Any unexpected signal patterns should be investigated for potential genetic rearrangements.

ZytoLight is a registered trademark of ZytoVision GmbH in Germany