Evaluation of a rapid method for extracting deoxyribonucleic acid (DNA)

摘要

Molecular laboratories require rapid and reliable methods for sample preparation. However, traditional DNA extraction techniques are generally time consuming (taking at least one hour to perform), and labor intensive (consisting of at least five manual steps). The purpose of this study was to compare a twostep DNA extraction method (GENERATION~(TM) Capture Disk Kit, Gentra, Minneapolis, MN) with our standard protocol (Puregene~R, Gentra, Minneapolis, MN). The Capture Disk method was used to extract DNA from buffy coat and plasma samples. Results were compared to DNA extractions performed using our standard protocol on the same samples, To determine if DNA could be extracted by the Capture Disk method, 30 human and two pig peripheral blood samples were amplified by the polymerase chain reaction (PCR) using primers for the human beta globin gene. To determine if the Capture Disk method could be utilized for an existing clinical test, 21 peripheral blood samples were extracted by both methods and then amplified using primers detecting a genetic mutation, factor V_(Leiden). Products from each PCR reaction were detected by gel electrophoresis. To determine the ability of the Capture Disk to isolate viral DNA, 20 plasma samples were extracted and amplified by PCR using primers for cytomegalovirus (CMV) and then detected colorimetrically. DNA bands were observed for all samples tested for human beta globin and factor V_(Leiden). There was 100% correlation of test results for factor V_(Leiden) between the two extraction methods. Isolation of viral DNA was unsuccessful. All known CMV positive samples tested were either negative or weakly positive. We also compared the time to perform each method as well as the associated labor and supply costs. The total extraction time for the GENERATION~(TM) Capture Disk averaged 26 minutes while the standard method took 102 minutes. The cost of the Capture Disk was dollar 1.40 per test compared to dollar 1,95 per test by the standard protocol, We conclude that the GENERATION~(TM) Capture Disk is a rapid technique for isolating DNA from clinical samples, It efficiently isolates DNA from buffy coats (as shown by the results for human beta globin) in sufficient quantities for clinical testing (as shown by the results for the factor V_(Leiden) test). Results were inconclusive for isolating viral DNA from plasma. The timesavings over the traditional technique is significant (opproximately 76 minutes per test). Cost is approximately dollar 0.55/test less than that incurred by the standard protocol,