How Much DNA is Lost? Measuring DNA Loss of STR Length Fragments Targeted by the PowerPlex 16® System Using the Qiagen MinElute Purification Kit

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

Clovis Age Western Stemmed Projectile Points and Human Coprolites at the Paisley Caves

The Paisley Caves in Oregon record the oldest directly dated human remains (DNA) in the Western Hemisphere. More than 100 high-precision radiocarbon dates show that deposits containing artifacts and coprolites ranging in age from 12,450 to 2295 ¹⁴C years ago are well stratified. Western Stemmed projectile points were recovered in deposits dated to 11,070 to 11,340 ¹⁴C years ago, a time contemporaneous with or preceding the Clovis technology. There is no evidence of diagnostic Clovis technology at the site. These two distinct technologies were parallel developments, not the product of a unilinear technological evolution. “Blind testing” analysis of coprolites by an independent laboratory confirms the presence of human DNA in specimens of pre-Clovis age. The colonization of the Americas involved multiple technologically divergent, and possibly genetically divergent, founding groups

Low wintertime vitamin D levels in a sample of healthy young adults of diverse ancestry living in the Toronto area: associations with vitamin D intake and skin pigmentation

<p>Abstract</p> <p>Background</p> <p>Vitamin D plays a critical role in bone metabolism and many cellular and immunological processes. Recent research indicates that concentrations of serum 25-hydroxyvitamin D [25(OH)D], the main indicator of vitamin D status, should be in excess of 75 nmol/L. Low levels of 25(OH)D have been associated with several chronic and infectious diseases. Previous studies have reported that many otherwise healthy adults of European ancestry living in Canada have low vitamin D concentrations during the wintertime. However, those of non-European ancestry are at a higher risk of having low vitamin D levels. The main goal of this study was to examine the vitamin D status and vitamin D intake of young Canadian adults of diverse ancestry during the winter months.</p> <p>Methods</p> <p>One hundred and seven (107) healthy young adults self-reporting their ancestry were recruited for this study. Each participant was tested for serum 25(OH)D concentrations and related biochemistry, skin pigmentation indices and basic anthropometric measures. A seven-day food diary was used to assess their vitamin D intake. An ANOVA was used to test for significant differences in the variables among groups of different ancestry. Linear regression was employed to assess the impact of relevant variables on serum 25(OH)D concentrations.</p> <p>Results</p> <p>More than 93% of the total sample had concentrations below 75 nmol/L. Almost three-quarters of the subjects had concentrations below 50 nmol/L. There were significant differences in serum 25(OH)D levels (p < 0.001) and vitamin D intake (p = 0.034) between population groups. Only the European group had a mean vitamin D intake exceeding the current Recommended Adequate Intake (RAI = 200 IU/day). Total vitamin D intake (from diet and supplements) was significantly associated with 25(OH)D levels (p < 0.001). Skin pigmentation, assessed by measuring skin melanin content, showed an inverse relationship with serum 25(OH)D (p = 0.033).</p> <p>Conclusion</p> <p>We observe that low vitamin D levels are more prevalent in our sample of young healthy adults than previously reported, particularly amongst those of non-European ancestry. Major factors influencing 25(OH)D levels were vitamin D intake and skin pigmentation. These data suggest a need to increase vitamin D intake either through improved fortification and/or supplementation.</p

To Clone or Not To Clone: Method Analysis for Retrieving Consensus Sequences In Ancient DNA Samples

The challenges associated with the retrieval and authentication of ancient DNA (aDNA) evidence are principally due to post-mortem damage which makes ancient samples particularly prone to contamination from “modern” DNA sources. The necessity for authentication of results has led many aDNA researchers to adopt methods considered to be “gold standards” in the field, including cloning aDNA amplicons as opposed to directly sequencing them. However, no standardized protocol has emerged regarding the necessary number of clones to sequence, how a consensus sequence is most appropriately derived, or how results should be reported in the literature. In addition, there has been no systematic demonstration of the degree to which direct sequences are affected by damage or whether direct sequencing would provide disparate results from a consensus of clones