A comparison of five methods for extracting DNA from paucicellular clinical samples.
Translational protocols in cancer and carcinogenesis often require isolation of genomic DNA from paucicellular clinical samples. DNA extraction methods for PCR-based applications should optimize the recovery of amplifiable DNA. We compared five methods for DNA extraction in paucicellular epithelial and lymphocyte samples using proportion of extractions producing amplifiable DNA and mean real-time PCR Ct values for GAPDH as the endpoint measures. The methods included solid-phase DNA adsorption (QIAamp), sequential protein and DNA precipitation (Puregene), magnetic bead adsorption (Dynabeads), phenol-chloroform extraction, and single-step proteinase K digestion. In general, the performance of the three commercial kits was superior to either phenol-chloroform extraction or single-step proteinase K digestion. However, QIAamp and Puregene produced amplifiable DNA more frequently than Dynabeads for starting cell numbers <50,000. GAPDH Ct values for QIAamp extractions showed the greatest dynamic range and the best linearity across the range of starting cell numbers, but QIAamp was not statistically significantly superior to Puregene. Of the three commercial kits, Puregene is the least expensive. QIAamp and Puregene DNA extraction methods are well-suited for the preparation of paucicellular clinical samples for PCR-based assays.
Cler
Leslie
L
Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, TX 75390-9155, USA.
Bu
Dawei
D
Lewis
Cheryl
C
Euhus
David
D
D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002725:Chloroform / Q000737:chemistry; D004247:DNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*; D016172:DNA Fingerprinting / Q000191:economics / Q000379:methods; D019286:Endopeptidase K / Q000737:chemistry / Q000191:economics; D006367:HeLa Cells; D006801:Humans; D019800:Phenol / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D011933:Reagent Kits, Diagnostic / Q000191:economics / Q000592:standards*; D015203:Reproducibility of Results
D011933:Reagent Kits, Diagnostic; D019800:Phenol; D002725:Chloroform; D004247:DNA; D019286:Endopeptidase K