Electrochemical detection of chromosomal translocation T (15;17) as an acute promyelotic leukemie marker
| Authors | |
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| Year of publication | 2003 |
| Type | Article in Proceedings |
| Conference | Sborník příspěvků, VII. pracovní setkání biochemiků a molekulárních biologů |
| MU Faculty or unit | |
| Citation | |
| Field | Electrochemistry |
| Keywords | Electrochemistry; chromosomal translocation T(15;17); promyelotic leukemie marker; PML/ RAR alfa translocation;DYNABEADS |
| Description | Acute myeloid leukemias are divided into several subtypes; one of them is the acute promyelocytic leukemia (APL) subtype M3 which is cytogenetically marked by the presence of t(15;17) translocation. This translocation involves the retinoic acid receptor alpha (RARa) gene on chromosome 17 and the PML locus on chromosome 15. Expression of the fusion gene produces a unique protein chimera PML/ RARa that is found in all APL casses. Various cytochemical, immunological and cytological methods have been used for precise analysis of APL. All these methods are suitable for diagnostics and typization of AML; however, their detection limit is relatively low - about one leukemic cell per 100 healthy ones. Molecular analyses of chromosomal abnormalities based on detection of specific mRNA and DNA molecules provide more sensitive tools for detection of AML. For example, reverse transcription polymerase chain reacion (RT-PCR) increases sensitivity of leukemic cell detection up to 10000-fold. This work describes a new approach for very sensitive and selective electrochemical detection of the unique fusion transcript PML/RARa. Total mRNA molecules purified from cell lysate are covalently bound to a target sequence (T25) on paramagnetic beads (DYNABEADS). To prove the presence of the unique transcript, a specific hybridization probe containing terminal sequence of 10 thymins was prepared. These thymins were modified with osmium as it is known that a suitable electrochemical probe is composed of the osmium complex as it preferentially binds to thymins. Unbound probe was removed with washing buffer, while the bound probe was released from the beads by temperature denaturation. The electrochemical detection of PML/RARa is in fact the usage of modified hybridization probe. Such a probe that hybridizes with a complementary strand can be easily and clearly detected using differential pulse stripping voltammetry on hanging mercury drop electrode. |
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