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A long distance-PCR derived FISH probe detects a deletion between p15 and p16 in CML and T-ALL patients
A Bleichert1, W Fiedler, U Claussen
1Institute of Human Genetics and Anthropology, Jena, Germany.
Abstract:
The tumor suppressor genes p15INK4B and p16INK4A, located in the chromosomal region 9p21, are frequently inactivated by homo- or hemizygous deletions, point mutation or promotor methylation in various types of cancer. No commercial probe is yet available that allows the detection of such deletions by FISH. Long distance (LD)-PCR was successfully used to generate a FISH probe, that covers a sequence stretch of 11.68 kb, located between the tumor suppressor genes p15 and p16. The LD-PCR amplicon was cloned and biotinylated by DOP-PCR (degenerated oligonucleotide primed-PCR) or nick translation. The FISH probe was hybridized on different samples of 16 patients with leukemia (3 T-ALL, 13 CML) and normal controls. Loss of at least one FISH-signal was found in 2/3 (67%) of the T-ALL- and 2/13 (15%) of the CML-cases. The new FISH probe presented here was proven to be advantageous for the detection of deletions in chromosomal region 9p21, especially between p15 and p16.
Insights
Researchers developed a new FISH probe to detect deletions in the 9p21 chromosomal region, crucial for tumor suppressor genes p15INK4B and p16INK4A. This probe successfully identified deletions in leukemia patients, aiding cancer diagnosis.
Area of Science:
- Genetics
- Molecular Biology
- Oncology
Background:
- Tumor suppressor genes p15INK4B and p16INK4A at chromosomal region 9p21 are frequently inactivated in cancers.
- Homo- or hemizygous deletions, point mutations, and promoter methylation are common inactivation mechanisms.
- Existing FISH probes do not adequately detect deletions between p15 and p16.
Purpose of the Study:
- To develop a novel FISH probe for detecting deletions in the 9p21 chromosomal region, specifically between the p15 and p16 genes.
- To evaluate the efficacy of the newly developed FISH probe in identifying deletions in leukemia patient samples.
Main Methods:
- Long-distance PCR (LD-PCR) was employed to generate a 11.68 kb FISH probe sequence between p15 and p16.
- The LD-PCR amplicon was cloned and biotinylated using degenerated oligonucleotide primed-PCR (DOP-PCR) or nick translation.
- FISH analysis was performed on samples from 16 leukemia patients (3 T-ALL, 13 CML) and normal controls.
Main Results:
- The new FISH probe successfully detected deletions in 67% of T-ALL cases (2/3) and 15% of CML cases (2/13).
- The probe demonstrated effectiveness in identifying deletions within the 9p21 region, specifically between the p15 and p16 genes.
- FISH signal loss correlated with the presence of deletions in the targeted chromosomal region.
Conclusions:
- A novel FISH probe has been successfully generated for detecting deletions between the p15 and p16 tumor suppressor genes.
- This probe is advantageous for identifying deletions in the 9p21 chromosomal region, particularly in leukemia.
- The developed FISH probe can aid in the diagnosis and understanding of cancers involving 9p21 deletions.