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

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. (J Mol Diagn 2010, 12:620 -628;

Liquid carry-over in an injection moulded all-polymer chip system for immiscible phase magnetic bead-based solid-phase extraction

“…2a shows the liquid carry-over of Milli-Q water as a function of MB mass and Table 1 shows the data needed to compare these results with already published work. As also reported by Sur et al [7], a linear correlation was found with a liquid carry-over of 1.74(8) nL/mg. MB extraction was not possible for less than 10 mg of MBs, because the magnetic force that could be applied to the MB cluster was insufficient to overcome the interfacial tension between the sample solution and the FC40 oil.…”

“…Sur et al used 40-180 mg of 450 nm MBs to estimate the carry-over of Tris buffer in their system amounting to 2.02 nL/mg. They also tested guanidinium isothiocyanate (GuSCN) and ethanol carry-over using 85 mg MBs [7]. Berry et al did not report details of their carry-over; however, they claim to be able to successfully extract 5.95 mg MBs in a 1% Triton X-100 (TX-100) PBS solution [4].…”

“…This is done by transporting the target-MB cluster through an immiscible phase, such as oil [4][5][6], wax [4,7,8], or air [8], often using an external permanent magnet. Such systems have been demonstrated for a number of applications including enzyme linked immunosorbent assays [6,9,10], nucleic acid extraction [4,7,8,[11][12][13][14], protein extraction [8], and cell extraction [15,16].…”

Microfluidic Sample Preparation for Medical Diagnostics

“…These devices do not require pumps or external hardware, except for a permanent magnet, and, therefore, may hold potential for purification technologies geared toward low-resource settings (62,63). Sur et al (64) developed a nucleic acid extraction cartridge with an automated magnetic mixer and mover to pull PMPs with adsorbed nucleic acid through liquid wax and into an elution chamber. Berry et al (65) based their immiscible filtration assisted by surface tension (IFAST) device on a similar principle, but added high-throughput capability to purify multiple messenger RNA samples in parallel.…”

Immiscible phase filter extraction and equivalent amplification of genotypes 1–6 of hepatitis C RNA: The building blocks for point-of-care diagnosis

“…Many commercial nucleic acid extraction systems process samples in a single well by repeatedly pelleting PMPs, aspirating the liquid, and adding the next solution. Numerous wash steps are often required to remove cell debris and other amplification inhibitors that adhere to tube surfaces, become entrapped in the magnetically-aggregated PMPs, or remain in the residual volume after the supernatant is removed by aspiration [20]. …”