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One-step purification of nucleic acid for gene expression analysis via Immiscible Filtration Assisted by Surface Tension (IFAST).

ABSTRACT: The extraction and purification of nucleic acids from complex samples (e.g. blood, biopsied tissue, cultured cells, food) is an essential prerequisite for many applications in biology including genotyping, transcriptional analysis, systems biology, epigenetic analysis, and virus/bacterial detection. In this report, we describe a new process of nucleic acid extraction that utilizes "pinned" aqueous/organic liquid interfaces in microchannels to streamline the extraction mechanism, replacing all washing steps with a single traverse of an immiscible fluid barrier, termed Immiscible Filtration Assisted by Surface Tension (IFAST). Nucleic acids in biological samples are bound to paramagnetic particles and then drawn across the IFAST device (or array of IFAST devices) using a magnet. While the strength of the IFAST barrier is suitable for separation of nucleic acids from lysate in its current embodiment, its permeability can be selectively adapted by adjusting the surface tensions/energies associated with the cell lysate, the immiscible phase, and the device surface, enabling future expansion to other non-nucleic acid applications. Importantly, processing time is reduced from 15-45 minutes to less than 5 minutes while maintaining purity, yield, and scalability equal to or better than prevailing methods. Operation is extremely simple and no additional lab infrastructure is required. The IFAST technology thus significantly enhances researchers' abilities to isolate and analyze nucleic acids, a process which is critical and ubiquitous in an extensive array of scientific fields.

SUBMITTER: Berry SM

PROVIDER: S-EPMC3244820 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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One-step purification of nucleic acid for gene expression analysis via Immiscible Filtration Assisted by Surface Tension (IFAST).

Berry Scott M SM Alarid Elaine T ET Beebe David J DJ

Lab on a chip 20110321 10

The extraction and purification of nucleic acids from complex samples (e.g. blood, biopsied tissue, cultured cells, food) is an essential prerequisite for many applications in biology including genotyping, transcriptional analysis, systems biology, epigenetic analysis, and virus/bacterial detection. In this report, we describe a new process of nucleic acid extraction that utilizes "pinned" aqueous/organic liquid interfaces in microchannels to streamline the extraction mechanism, replacing all wa ...[more]

PMID: 21423999

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Project description:BackgroundDewatering constitutes a major challenge to the production of microalgae, accounting for 20-30% of the product cost. This presents a setback for the applicability of microalgae in the development of several sustainable products. This study presents an investigation into the dynamic dewatering of microalgae in a combined flocculation-filtration process. The effect of process conditions on the performance of 12 flocculants and their mixtures was assessed.ResultsThe mechanism of flocculation via the electrostatic path was dominated by charge neutralization and subsequently followed bridging in a 'sweep flocculation' process. Cationic polyacrylamide (CPAM) based flocculants recorded the highest biomass retention with PAM1 and PAM2 attaining 99 and 98% retention with flocculant dosages of 10 and 15 mg/L respectively. Polyvinylamine (PVAM) was also found to improve system stability across the pH range 4-10. Alum was observed to be only effective in charge neutralization, bringing the system close to its isoelectric point (IEP). Chemometric analysis using the multi-criteria decision methods, PROMETHEE and GAIA, was applied to provide a sequential performance ranking based on the net outranking flow (ф) from 207 observations. A graphical exploration of the flocculant performance pattern, grouping the observations into clusters in relation to the decision axis ([Formula: see text]), which indicated the weighted resultant of most favorable performance for all criteria was explored.ConclusionCPAM based flocculants and their mixtures demonstrated superior performance due to their viscoelastic behaviour under turbulence. The use of PVAM or alum in mixtures with CPAM reduced the required doses of both flocculants, which will provide beneficial financial impact for largescale microalgae dewatering in a flocculant assisted dynamic filtration process. Chemometric analysis based on the physico-chemical properties of the system provides a time saving assessment of performance across several criteria. The study findings provide an important foundation for flocculant assisted dynamic filtration processes.

Dataset Information

One-step purification of nucleic acid for gene expression analysis via Immiscible Filtration Assisted by Surface Tension (IFAST).

Publications

One-step purification of nucleic acid for gene expression analysis via Immiscible Filtration Assisted by Surface Tension (IFAST).

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