Open Access Preprint
Final Published Version
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 performed 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 (qPCR) 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 106–409 base pairs (bps) in length, corresponding to those targeted by the Promega PowerPlex 16® System. Across all standards starting from 10^4 to 10^7 copies/μL, loss averaged between 21.75% to 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 between neither DNA strand length and retention, nor 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 how their claims are made, 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.
Kemp, Brian M.; Winters, Misa; Monroe, Cara; and Barta, Jodi Lynn, "How Much DNA is Lost? Measuring DNA Loss of STR Length Fragments Targeted by the PowerPlex 16® System Using the Qiagen MinElute Purification Kit" (2014). Human Biology Open Access Pre-Prints. 61.