title: How much DNA is lost? Measuring DNA loss of short-tandem-repeat length fragments targeted by the PowerPlex 16® system using the Qiagen MinElute Purification Kit.
abstract: The success in recovering genetic profiles from aged and degraded biological samples is diminished by fundamental aspects of DNA extraction, as well as its long-term preservation, that are not well understood. While numerous studies have been conducted to determine whether one extraction method was superior to others, nearly all of them were initiated with no knowledge of the actual starting DNA quantity in the samples prior to extraction, so they ultimately compared the outcome of all methods relative to the best. Using quantitative PCR to estimate the copy count of synthetic standards before (i.e., "copies in") and after (i.e., "copies out") purification by the Qiagen MinElute PCR Purification Kit, we documented DNA loss within a pool of 16 different-sized fragments ranging from 106 to 409 bp in length, corresponding to those targeted by the PowerPlex 16 System (Promega, Madison, WI). Across all standards from 10(4) to 10(7) copies/μL, loss averaged between 21.75% and 60.56% (mean, 39.03%), which is not congruent with Qiagen's claim that 80% of 70 bp to 4 kb fragments are retained using this product (i.e., 20% loss). Our study also found no clear relationship either between DNA strand length and retention or between starting copy number and retention. This suggests that there is no molecule bias across the MinElute column membrane and highlights the need for manufacturers to clearly and accurately describe on what their claims are based, and should also encourage researchers to document DNA retention efficiencies of their own methods and protocols. Understanding how and where to reduce loss of molecules during extraction and purification will serve to generate clearer and more accurate data, which will enhance the utility of ancient and low-copy-number DNA as a tool for closing forensic cases or in reconstructing the evolutionary history of humans and other organisms.
authors.affiliation: 1 Department of Anthropology, Washington State University, Pullman, Washington.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Kemp
authors.forename: Misa
authors.initials: M
authors.lastname: Winters
authors.forename: Cara
authors.initials: C
authors.lastname: Monroe
authors.forename: Jodi Lynn
authors.initials: JL
authors.lastname: Barta
mesh_terms: D016172:DNA Fingerprinting / Q000295:instrumentation* / Q000379:methods; D005819:Genetic Markers / Q000235:genetics*; D005820:Genetic Testing; D005838:Genotype; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012015:Reference Standards
chemical_list: D005819:Genetic Markers
keywords: dna extraction; dna loss; dna standards; extraction efficiency; promega powerplex 16® system; qiagen minelute