Immiscible Phase Nucleic Acid Purification Eliminates PCR Inhibitors With a Single Pass of Paramagnetic Particles Through a Hydrophobic Liquid

J Mol Diagn. 2010 Sep;12(5):620-8. doi: 10.2353/jmoldx.2010.090190. Epub 2010 Jun 25.

Abstract

Extraction and purification of nucleic acids from complex biological samples for PCR are critical steps because inhibitors must be removed that can affect reaction efficiency and the accuracy of results. This preanalytical processing generally involves capturing nucleic acids on microparticles that are then washed with a series of buffers to desorb and dilute out interfering substances. We have developed a novel purification method that replaces multiple wash steps with a single pass of paramagnetic particles (PMPs) though an immiscible hydrophobic liquid. Only two aqueous solutions are required: a lysis buffer, in which nucleic acids are captured on PMPs, and an elution buffer, in which they are released for amplification. The PMPs containing the nucleic acids are magnetically transported through a channel containing liquid wax that connects the lysis chamber to the elution chamber in a specially designed cartridge. Transporting PMPs through the immiscible phase yielded DNA and RNA as pure as that obtained after extensive wash steps required by comparable purification methods. Our immiscible-phase process has been applied to targets in whole blood, plasma, and urine and will enable the development of faster and simpler purification systems.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chlamydia Infections / diagnosis
  • Chlamydia Infections / microbiology
  • Chlamydia trachomatis / genetics
  • Chlamydia trachomatis / isolation & purification
  • Gonorrhea / diagnosis
  • HIV Infections / diagnosis
  • HIV-1 / genetics
  • HIV-1 / isolation & purification
  • Humans
  • Neisseria gonorrhoeae / genetics
  • Neisseria gonorrhoeae / isolation & purification
  • Nucleic Acids / isolation & purification*
  • Reverse Transcriptase Polymerase Chain Reaction*

Substances

  • Nucleic Acids