Abstract
The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions.
Highlights
The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging
Escherichia coli DNA was extracted from an NEB 5-alpha strain using Epicentre QuickExtract DNA Extraction Buffer (Lucigen Corporation,Middleton, WI, USA) and the stock was quantified at 1.4 × 107 cp/μL using digital PCR (dPCR)
We mixed the kit extract into a qPCR reaction spiked with λ phage DNA at either a 10x dilution (1 μL kit extract, 0.5 μL template DNA, 8.5 μL reaction mix) or 2.5x dilution (4 μL kit extract, 0.5 μL template, 5.5 μL reaction mix)
Summary
The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. The resulting eluent contains NAs and carryover of extraction buffers These inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 μL eluent in 9 μL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. We first identified that kit buffer carryover is a concern when using low eluent dilutions (≤2.5x) for both commercial silica-column and magnetic-bead extractions (following manufacturer protocols). To unambiguously show that inhibition is due to kit buffer inhibitors, as opposed to sample inhibitors or losses of NAs, we performed extractions on pure water samples with or without the TPW, and added the resulting kit extract to spiked qPCR, LAMP, and RT assays
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