Nucleic acid sample preparation using spontaneous biphasic plug flow.
Nucleic acid (NA) extraction and purification has become a common technique in both research and clinical laboratories. Current methods require repetitive wash steps with a pipet that are laborious and time-consuming, making the procedure inefficient for clinical settings. We present here a simple technique that relies on spontaneous biphasic plug flow inside a capillary to achieve sample preparation. By filling the sample with oil, aqueous contaminants were displaced from the captured NA without pipetting wash buffers or use of external force and equipment. mRNA from mammalian cell culture was purified, and polymerase chain reaction (PCR) amplification showed similar threshold cycle values as those obtained from a commercially available kit. Human immunodeficiency virus (HIV) viral-like particles were spiked into serum and a 5-fold increase in viral RNA extraction yield was achieved compared to the conventional wash method. In addition, viral RNA was successfully purified from human whole blood, and a limit of detection of approximately 14 copies of RNA extracted per sample was determined. The results demonstrate the utility of the current technique for nucleic acid purification for clinical purposes, and the overall approach provides a potential method to implement nucleic acid testing in low-resource settings.
Department of Biomedical Engineering and Wisconsin Institute for Medical Research, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
Peter C
PC
Thomas
Lindsay N
LN
Strotman
Ashleigh B
AB
Theberge
Erwin
E
Berthier
Rachel
R
O'Connell
Jennifer M
JM
Loeb
Scott M
SM
Berry
David J
DJ
Beebe
D000818:Animals; D002197:Capillary Action; D002417:Cattle; D057809:HEK293 Cells; D015497:HIV-1; D006801:Humans; D061986:MCF-7 Cells; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009696:Nucleic Acids / Q000032:analysis*
D009696:Nucleic Acids