title: Modification of standard proteinase K/phenol method for DNA isolation to improve yield and purity from frozen blood.
abstract: Research in medical genetics may frequently involve freezing of large numbers of peripheral blood samples. This is a convenient method for storing blood for subsequent DNA isolation and analysis. An area of potential concern is the low yield of DNA from blood samples that have been frozen. Here we report a modification of the widely used standard proteinase K/phenol DNA isolation method for improving the yield and purity of DNA from frozen blood samples, by an initial trypsinisation of whole blood before cell lysis to obtain lymphocytic nuclei and subsequent DNA purification. We report an increased total yield of DNA with pretrypsinised blood as well as improved purity. These results indicate that trypsinisation of thawed whole blood helps the deproteinisation process, reducing the amount of protein associated with the nuclear pellet. This modification to improve yield and purity of DNA from frozen blood samples should be useful to laboratories performing DNA based diagnostic work or studying molecular genetic mechanisms of disease.
authors.affiliation: Research Department, Wills Eye Hospital, Philadelphia, PA 19107, USA.
authors.forename: N N
authors.initials: NN
authors.lastname: Ahmad
authors.forename: A B
authors.initials: AB
authors.lastname: Cu-Unjieng
authors.forename: L A
authors.initials: LA
authors.lastname: Donoso
mesh_terms: D001793:Blood Preservation; D015925:Cryopreservation; D004247:DNA / Q000097:blood* / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D005821:Genetic Techniques; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D019800:Phenol; D010636:Phenols; D012697:Serine Endopeptidases; D014357:Trypsin
chemical_list: D010636:Phenols; D019800:Phenol; D004247:DNA; D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K