Immiscible phase nucleic acid purification eliminates PCR inhibitors with a single pass of paramagnetic particles through a hydrophobic liquid.
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.
Sur
Kunal
K
Center for Innovation in Global Health Technologies, Northwestern University, Evanston, Illinois 60208, USA.
McFall
Sally M
SM
Yeh
Emilie T
ET
Jangam
Sujit R
SR
Hayden
Mark A
MA
Stroupe
Stephen D
SD
Kelso
David M
DM
D002690:Chlamydia Infections / Q000175:diagnosis / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000235:genetics / Q000302:isolation & purification; D006069:Gonorrhea / Q000175:diagnosis; D015658:HIV Infections / Q000175:diagnosis; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D009344:Neisseria gonorrhoeae / Q000235:genetics / Q000302:isolation & purification; D009696:Nucleic Acids / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D009696:Nucleic Acids