Rapid DNA extraction and sensitive alkaline blotting protocol: application for detection of gene rearrangement and amplification for clinical molecular diagnosis.
A simple and sensitive DNA extraction/Southern blotting method is described for the routine detection of gene rearrangement and amplification in DNA samples. Significant features of the procedure are: (1) sequential digestion of RNA and protein in the same DNA buffer; and (2) use of a modified alkaline transfer protocol which increases sensitivity and decreases the autoradiographic exposure period. The method lends itself to handling multiple samples containing minimal amounts of starting material, requires equipment commonly found in a clinical laboratory, and yields results within 36 h of specimen receipt. Accordingly, these procedures may find utility in the establishment of molecular diagnostics in a conventional clinical setting.
Fourney
R M
RM
Molecular Genetics and Carcinogenesis Laboratory, Cross Cancer Institute, Edmonton, Alberta, Canada.
Dietrich
K D
KD
Paterson
M C
MC
D015139:Blotting, Southern / Q000379:methods; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D015342:DNA Probes / Q000302:isolation & purification; D005784:Gene Amplification; D015321:Gene Rearrangement; D006801:Humans; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length
D015342:DNA Probes; D004247:DNA