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

Wheeler, E K ; Erler, A M ; Seiler, A

关键词: DETECTION;    DNA;    ETHANOL;    LOSSES;    MASS BALANCE;    PACKED BEDS;    POLYMERASE CHAIN REACTION;    PRECIPITATION;    PROCESSING;    TRACE AMOUNTS;   

DOI  :  10.2172/929531
RP-ID  :  LLNL-TR-403178
PID  :  OSTI ID: 929531
Others  :  TRN: US200822%%988

美国|英语

来源: SciTech Connect

【 摘 要 】

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.