Functionalized nanoporous particles allow single-step purification of DNA reactions
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Functionalized nanoporous particles allow single-step purification of DNA reactions The pitch Using a new materials-based approach, Diffinity Genomics is introducing a series of products for rapid, single-step purification of nucleic acid reactions such as polymerase chain reaction (PCR) amplification of DNA. The initial product, the Diffinity RapidTipTM for PCR Purification, removes unwanted materials from the PCR solution such as unincorporated oligonucleotides used as primers plus nucleotides (dNTP) thereby rendering the solution suitable for DNA sequencing reactions. Currently, PCR purification is done either by enzymes that selectively degrade the unwanted components (enzymatic cleanup) or by using differential solubility to selectively elute the desired double strands after immobilizing all of the nucleic acids (bind-wash-elute cleanup). Each of these processes takes 10–30 minutes and involves the addition of reagents. The approach taken by Diffinity Genomics is based on a single step in which the post-PCR solution is aspirated into a pipette tip containing functional material and the purified product dispensed. It is quicker than existing processes (60 seconds versus 10–30 minutes) and less
labor intensive. In addition, the need for reagents, additional consumables, or other infrastructure such as centrifuges, vacuum manifolds, or magnetic handlers is eliminated. The current market for PCR cleanup products is over $200 million per year. The customers are the approximately 400,000 researchers and laboratory technicians worldwide who study genomics or use it in their work. Diffinity’s initial product, RapidTipTM, improves workflow, is easier to use, and reduces operator-related errors thereby saving substantial time and labor costs. There is estimated to be a sizable number of researchers who currently use “home brew” PCR purification methods because of the expense and inconvenience of cleanup products.
The technology
The materials strategy enabling singlestep rapid purification is shown schematically in the figure where nanoporous particles can be functionalized so that small and large molecules can experience different surface adsorption characteristics. For this specific application, the pore interiors are selectively functionalized so that only the primers and unincorporated nucleotides in PCR product solutions can penetrate to be highly adsorbing Add PCR while separately treatproduct solution ing the particle exteriors experienced by the DNA amplicons (piecSurface coating that captures dNTP and ss-DNA es of DNA formed by Surface coating that blocks amplicon adsorption natural or synthetic Unamplified primers Unincorporated dNTP amplification) to have PCR Amplicons distinct properties. This combination of Schematic depiction of differentially functionalized nanoposize-exclusion chrorous particles for purification of polymerase chain reaction products. matography with differential functionaliza-
tion allows the rapid selective adsorption of the impurities while retaining the desired DNA in solution when t
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