title: The 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.
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. 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. Methods investigated included both liquid/liquid (phenol-chloroform) and solid phase based robotic procedures, (PrepFiler™ 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 suggests 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.
authors.affiliation: Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA.
authors.forename: Lilliana I
authors.initials: LI
authors.lastname: Moreno
authors.affiliation: Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA. mccordb@fiu.edu.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: McCord
mesh_terms: D001647:Bile Acids and Salts; D004247:DNA / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification*; D004492:Edetic Acid; D006801:Humans; D013058:Mass Spectrometry; D008543:Melanins; D018895:Microsatellite Repeats / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019800:Phenol; D013997:Time Factors
chemical_list: D001647:Bile Acids and Salts; D008543:Melanins; D019800:Phenol; D004247:DNA; D004492:Edetic Acid
keywords: DNA inhibitors; DNA purification; Direct analysis in real-time (DART)