Physical lysis only (PLO) methods suitable as rapid sample pretreatment for qPCR assay

Xiaofang Wang, Byung Tae Lee, Ahjeong Son

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Quantitative PCR (qPCR) enables rapid and sensitive gene quantification and is widely used in genomics, such as biological, medical, environmental, and food sciences. However, sample pretreatment requires the use of conventional DNA extraction kits which are time-consuming and labor intensive. In this study, we investigated four physical lysis only (PLO) methods which are rapid and could serve as alternatives to conventional DNA extraction kits. These PLO methods are bead mill, heating, sonication, and freeze–thaw. Using ethidium bromide-based assay, their performance was evaluated and compared. The effects of cell debris and its removal were also investigated. Bead mill method without cell debris removal appeared to yield the best qPCR results among the four PLO methods. In addition, bead mill method also performed better than conventional DNA extraction kits. It is probably due to the substantial loss of DNA material during the extensive purification of the conventional DNA extraction kits. The bead mill method has been demonstrated to successfully quantify 102 to 107 copies of the PAH-RHDα gene of Pseudomonas putida.

Original languageEnglish
Pages (from-to)8719-8728
Number of pages10
JournalApplied Microbiology and Biotechnology
Volume98
Issue number20
DOIs
StatePublished - Oct 2014

Bibliographical note

Funding Information:
Acknowledgments This work was supported by National Science Foundation (CAREER award #1054768) and Korean National Research Foundation (#2014003129).

Publisher Copyright:
© 2014, Springer-Verlag Berlin Heidelberg.

Keywords

  • Cell lysis
  • DNA extraction
  • Pseudomonas putida
  • Quantitative PCR

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