Isolation and characterization of human DNA in agarose block.
Classic phenol-chloroform extraction of human DNA results in substantial shearing and low yield. Because DNA analysis in human genetic disorders needs relatively intact DNA, we modified the existing method and systematically analyzed the human DNA isolated from HeLa cells, leukocytes, amniocytes, and fibroblasts in agarose block and compared the results to those obtained by conventional phenol method. Our results showed that DNA isolated by the agarose method was higher in molecular weight, with minimal shearing as compared to the phenol method. Yield of DNA from the agarose method was substantially higher, almost twice that obtained by the phenol method. Restriction enzyme digestion of DNA from the agarose method indicated the usefulness of this DNA for restriction fragment length polymorphism (RFLP) analysis without further purification. DNA obtained by the agarose method was found to be more resistant to thermal degradation and more stable on long-term storage than that of phenol-extracted DNA.
Yanamandra
G
G
Department of Pediatrics, UMDNJ-Robert Wood Johnson Medical School, New Brunswick 08903.
Lee
M L
ML
D000650:Amnion / Q000166:cytology; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D005347:Fibroblasts / Q000166:cytology; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D007962:Leukocytes / Q000166:cytology; D008970:Molecular Weight; D011309:Preservation, Biological / Q000379:methods; D012685:Sepharose
D004247:DNA; D012685:Sepharose