Single copy gene detection requiring minimal cell numbers.
There has been an increasing application of molecular DNA probes to evaluate a variety of clinical conditions. Frequently, the amount of tissue or number of cells available limits analysis by conventional DNA extraction and Southern blot hybridization. Moreover, DNA amplification techniques cannot be used in all cases. We have applied a modification of the DNA extraction-Southern blot hybridization technique to clinical samples which provides essentially quantitative recovery and analysis of DNA from minimal numbers of cells. DNA was obtained from cells which were immobilized in agarose blocks for lysis, deproteinization and restriction enzyme digestion. The DNA was then run directly into agarose gels to size fractionate for Southern blot analysis. Cells can be suspended in agarose blocks for over one year and frozen cells can be thawed and suspended in agarose. A variety of restriction enzymes can be used. Single copy sequences can be detected from as few as 5 x 10(4) cells. We have employed this method to examine immunoglobulin gene rearrangements in PBL from leukemia patients as well as bone marrow from myeloma patients. In addition, we have used the technique to accurately assess bone marrow engraftment after transplant. These results demonstrate a diagnostic application of this technique in a variety of clinical samples where there may be limited availability of cells.
Feddersen
R M
RM
University of Minnesota.
Van Ness
B G
BG
D000818:Animals; D001402:B-Lymphocytes / Q000032:analysis; D001709:Biotechnology; D015139:Blotting, Southern; D016026:Bone Marrow Transplantation; D002452:Cell Count; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D005784:Gene Amplification; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics; D009101:Multiple Myeloma / Q000235:genetics
D015342:DNA Probes; D004247:DNA