c-kit mutation screening in patients with acute myeloid leukaemia: adaptation of a Giemsa-stained bone-marrow smear DNA extraction technique

Citation
El. Court et al., c-kit mutation screening in patients with acute myeloid leukaemia: adaptation of a Giemsa-stained bone-marrow smear DNA extraction technique, BR J BIOMED, 58(2), 2001, pp. 76-84
Citations number
14
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Medical Research Diagnosis & Treatment
Journal title
BRITISH JOURNAL OF BIOMEDICAL SCIENCE
ISSN journal
0967-4845 → ACNP
Volume
58
Issue
2
Year of publication
2001
Pages
76 - 84
Database
ISI
SICI code
0967-4845(2001)58:2<76:CMSIPW>2.0.ZU;2-2
Abstract
The scarcity of viable tissue samples for leukaemia research is widely reco gnised and currently restrictive. Archival bone-marrow smears pre sent a va luable resource that can be exploited easily for mutational analysis. Here, a modified technique to extract DNA is described, and used subsequently fo r mutation/polymorphism screening of the stem-cell factor receptor protoonc ogene c-kit in 23 patients with acute myeloid leukaemia (AML). The selected method was straightforward and used bone-marrow material scraped from peri odic acid-Schiff, sudan black B and May-Grunwald/Giemsa-stained preparation s, and treated initially with proteinase K prepared in digestion buffer to digest all proteinaceous matter. Following incubation, saturated sodium chl oride was added and DNA extracted from the supernatant by phenol/chloroform /isoamyl alcohol treatment. Retrieved DNA was precipitated with ethanol at -20 degreesC overnight, washed with 95% ethanol, air-dried, resuspended usi ng purite water and stored at -20 degreesC prior to use in mutational analy sis. The extraction method described was compared with a commercial reagent for combined DNA, RNA and protein isolation using cryopreserved cells from 20 patients with AML. The quality of extracted DNA isolated by the two met hods was comparable by polymerase chain reaction (PCR) and single-strand co nformation polymorphism (SSCP) techniques. Bone-marrow biopsies are perform ed regularly on each AML patient to monitor the disease; therefore, an extr action method using this resource could liberate a valuable source of DNA f or study (e.g. molecular investigations, including mutation/polymorphism sc reening etc.). This would allow fresh and programme-frozen cells to be rese rved for those investigations requiring intact, viable cells. The use of ar chived bone-marrow smears would permit vast increase in the: scope for retr ospective testing and large-scale analyses.