C-kit mutation screening in patients with acute myeloid leukaemia: adaptation of a Giemsa-stained bone-marrow smear DNA extraction technique.
The scarcity of viable tissue samples for leukaemia research is widely recognised and currently restrictive. Archival bone-marrow smears present a valuable resource that can be exploited easily for mutational analysis. Here, a modified technique to extract DNA is described, and used subsequently for mutation/polymorphism screening of the stem-cell factor receptor proto-oncogene c-kit in 23 patients with acute myeloid leukaemia (AML). The selected method was straightforward and used bone-marrow material scraped from periodic acid-Schiff, sudan black B and May-Grünwald/Giemsa-stained preparations, and treated initially with proteinase K prepared in digestion buffer to digest all proteinaceous matter. Following incubation, saturated sodium chloride was added and DNA extracted from the supernatant by phenol/chloroform/isoamyl alcohol treatment. Retrieved DNA was precipitated with ethanol at -20 degrees C overnight, washed with 95% ethanol, air-dried, resuspended using purite water and stored at -20 degrees C prior to use in mutational analysis. 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 methods was comparable by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) techniques. Bone-marrow biopsies are performed regularly on each AML patient to monitor the disease; therefore, an extraction method using this resource could liberate a valuable source of DNA for study (e.g. molecular investigations, including mutation/polymorphism screening etc.). This would allow fresh and programme-frozen cells to be reserved for those investigations requiring intact, viable cells. The use of archived bone-marrow smears would permit vast increase in the scope for retrospective testing and large-scale analyses.
Court
E L
EL
Centre for Research in Biomedicine, University of the West of England, Bristol, UK.
Davidson
K
K
Smith
M A
MA
Inman
L
L
Marriott
S A
SA
Smith
J G
JG
Pallister
C J
CJ
D000208:Acute Disease; D001854:Bone Marrow Cells / Q000737:chemistry*; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D000090063:Proto-Oncogene Mas; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*
D004273:DNA, Neoplasm; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D019009:Proto-Oncogene Proteins c-kit