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Report on Qiagen Columns with Precipitation versus Packed Bed Technology for Trace Amounts of DNA

Description: The assured limit of detection (LOD), where 100% of the PCR assays are successful, for the Qiagen spin column is dramatically improved when combined with an ethanol precipitation step of the eluted sample. A detailed SOP for the ethanol precipitation was delivered as a separate report. A key finding in the precipitation work was to incubate the ethanol precipitation at -20{sup o}C overnight when concentrating low copy number samples. Combining this modified ethanol precipitation with the Qiagen spin columns, the limit of assured detection was improved by 1-2 orders of magnitude, for the aliquot and assay variables used. The lower limit of detection (defined as when at least 1 assay of 1 aliquot was positive) was only improved by approximately 1 order of magnitude. The packed bed process has the potential of a 20-fold improvement in the limit of detection compared to Qiagen plus precipitation, based on a mass balance analysis for the entire DNA concentration and purification processes. Figure ES1 shows a mass balance for all the DNA processing steps. The packed bed process minimizes losses from elution, precipitation, and pipetting (aliquoting and transferring). Figure ES1 assumes that 100 copies of DNA serve as the input sample. Efficiencies for each step have been estimated based on our experiences or a worst case scenario (for example, a 50% loss was assumed for pipetting). Table ES1 summarizes the number of copies that are the input template for PCR assuming 100 copies of DNA are processed through the three options detailed in Figure ES1.Theoretically a 20-fold increase in the number of starting copies in the PCR reaction is gained when the DNA is concentrated, purified and then amplified directly on the surface of the beads in the packed bed.
Date: February 5, 2008
Creator: Wheeler, E K; Erler, A M & Seiler, A
Partner: UNT Libraries Government Documents Department

Comparison of Packed Beds and Qiagen Columns for Recovering Trace Amounts of B. anthracis DNA from Liquid Suspensions

Description: The goal of this work was to optimize and evaluate LLNL's in-bed amplification technology to improve the level of detection for suspensions containing trace amounts of anthracis DNA. The binding/cleaning performance of the packed bed is compared to the conventional commercial approach; Qiagen column cleanup and elution, followed by detection through an ex-situ amplification process. Five liquid suspensions were spiked with B.anthracis DNA in concentration series. These suspensions were: (1) water, (2) water with EDTA, (3) dirty water from carpet extraction, (4) dirty carpet extraction with phosphate buffered saline (PBS) plus 0.1% Tween 20 plus 0.1% gelatin, and (5) a subway aerosol collected in water. Each suspension matrix was spiked with DNA and injected (in replicate) into either Qiagen Microcolumns (using the kit processing instructions) or LLNL's packed bed (using the LLNL in-bed purification and amplification protocol). The process output was assayed by quantitative polymerase chain reaction (QPCR). Table ES-1 shows the level of DNA (pg per 100 uL of input suspension) that resulted in successful amplification for all reactions (X=Y), and the level for which at least one of the reactions was successful (X>0). For each suspension and DNA concentration, there were Y QPCR assays of which X showed successful amplification. LLNL's packed bed technology outperformed Qiagen Microcolumns for all five suspensions, typically by one order of magnitude in both the limit of assured detection (all reactions positive), and the lower limit of detection (some reactions positive).
Date: June 23, 2006
Creator: Sorensen, K; Arroyo, E; Erler, A; Christian, A T; Camp, D & Wheeler, E K
Partner: UNT Libraries Government Documents Department