A Parametric Study of Sample Lysis and DNA Purification Techniques for Use in Automated Devices

Abstract

We present a parametric study on the efficiency of several automatable procedures for the extraction and purification of DNA from a variety of pathogens. Based on the results of this work, an optimized protocol has been developed for use with both spiked buffers and nasal wash. All steps of this protocol are suitable for incorporation into a field-portable, automated sample preparation device. From introduction of the sample to elution of DNA, the entire process was completed in less than 60 min, and the time could be reduced further by automation. The recovered DNA is of sufficient quality for real-time or multiplex PCR amplification. The protocol was demonstrated on nasal wash samples spiked with E. coli containing the J7R and crmB genes. Subsequent testing on resequencing microarrays correctly identified the samples as Variola major virus.

Keywords:

• Automatable
• DNA purification
• lysis
• sample preparation
• solid phase extraction

ACKNOWLEDGMENTS

JSE is a National Research Council Postdoctoral Fellow. This research was supported by NRL 5.2 work unit 6006. The opinions and assertions contained herein are those of the authors and are not to be construed as those of the U.S. Navy or military service at large.

Notes

Treatments: QB, Qiagen buffer PB; DMSO, 10 vol% DMSO in QB; Lysozyme, 1 mg/mL lysozyme for 30 min and then QB added and incubated another 30 min; SDS, 0.1 wt% SDS in QB. Samples (except lysozyme) were each incubated in lysis buffer for 30 min before sonication. Sonicated samples were first subjected to chemical treatment and then sonicated for 1 min at 65 W.

Resequencing chips allows base-by-base comparison between the sample DNA and reference sequences, in this case VMV genes. Analysis software then looks at the quantity and pattern of identical bases to determine if the sample DNA is sufficiently similar to the reference to make a positive identification. In all experiments, the high percentage of matched bases provided enough sequence information to identify the genes and the organism.