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                            Library & Multimedia
 Use of Direct Analysis in Real Time (DART) To Assess the Levels of Inhibitors Co-extracted With DNA and the Associated Impact in Quantification and Amplification
NCJ Number
              251255

Author(s)
          L. I. Moreno, B. R. McCord
Date Published

June 2016
Length
              10 pages
Annotation
              Direct analysis in real time (DART) mass spectrometry was used in this study to investigate the ability of different extraction methods to remove PCR inhibitors.
Abstract
              The measure of quality in DNA sample processing starts with an effective nucleic acid isolation procedure. Most problems with DNA sample typing can be attributed to low quantity DNA and/or to the presence of inhibitors in the sample; therefore, establishing which isolation method is best at removing potential inhibitors may help overcome some of the problems analysts encounter by providing useful information in the determination of the optimal approach for any given sample. In the current study, methods investigated included both liquid/liquid (phenol–chloroform) and solid phase based robotic procedures, (PrepFilerTM and EZ1 chemistries). Following extraction, samples were analyzed by DART in order to determine the level of remaining inhibitors and then quantified and amplified to determine the effect any remaining inhibitor had on the overall results. The data suggest that organic extraction methods result in detrimental amounts of phenol carryover while automated methods may produce carry-over of bile salts and other chemicals that preferentially bind the solid phase matrix. Both of these effects can have a negative impact in downstream sample processing and genotyping by PCR. (Publisher abstract modified)
      
  
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        Detection and remediation of PCR inhibition using real time PCR melt curves as a diagnostic tool.
    
  
  
Date Published: June 1, 2016
    
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