Isotachophoresis of nucleic acids in agarose gel rods

Abstract

A new method of electrophoresis (isotachophoresis in agarose gel rods) in which nucleic acid molecules are not separated but, oppositely, are brought together into one band, was elaborated. Heterogeneous in size DNA and RNA polymers present in a few milliliters of a solution at so low concentration that their isolation by other methods is hardly attainable and fraught with losses are brought together into one visible narrow band when put in a discontinuous electric field. Polynucleotides migrate in dilute (0.1%) semifluid agarose gel that permits easy quantitative isolation of the band of interest. Resulting DNA can be used directly in PCR. The suggested method for isolation of micro amounts of nucleic acids from dilute solutions can be applied to forensic and clinical research and cancer gene diagnostics by the analysis of fragmented circulating DNA from bodily fluids.

Isotachophoresis: Theory and Microfluidic Applications

“…Consider that protein mobility, function, and solubility are all a strong function of pH. For example, proteins are often focused or separated using cationic ITP, since many proteins have a positive charge in electrolytes buffered near pH 6–8 (i.e., relatively high isoelectric points, p I values). − Anionic ITP is typically the most useful mode for assays involving the focusing and separation of nucleic acids (NAs, as in DNA or RNA) and negatively charged proteins (i.e., proteins with relatively high p I values). ,− NAs typically remain negatively charged over a broad pH range (with a p K a of ∼1.5 due to the phosphate backbone), although nucleic acid solutions should be pH-buffered for stability and function.…”

HIV detection from human serum with paper-based isotachophoretic RNA extraction and reverse transcription recombinase polymerase amplification

“…Kondratova et al were the first to use ITP in agarose gels for DNA extraction from human blood samples, but their work was not wellsuited for POC diagnostics because it required lengthy deproteinization and dialysis pretreatment steps. 14,17 Microchannel-based ITP has since emerged as a promising sample preparation approach for extracting DNA from blood specimens and amplifying with off-chip quantitative polymerase chain reaction (qPCR). [18][19][20][21] Notably, Eid et al detected DNA from Listeria monocytogenes cells in 2.5 µL of whole blood using alkaline and proteinase K lysis, microchannel ITP purification, and recombinase polymerase amplification for detection.…”

HIV Detection from Human Serum with Paper-Based Isotachophoretic RNA Extraction and Reverse Transcription Recombinase Polymerase Amplification

“…Kondratova et al were the first to use ITP in agarose gels for DNA extraction from human blood samples, but their work was not wellsuited for POC diagnostics because it required lengthy deproteinization and dialysis pretreatment steps. 14,17 Microchannel-based ITP has since emerged as a promising sample preparation approach for extracting DNA from blood specimens and amplifying with off-chip quantitative polymerase chain reaction (qPCR). [18][19][20][21] Notably, Eid et al…”