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In this study, we sought to document the efficiency of primer extension capture (PEC) as a method to enrich DNA eluates of targeted DNA molecules and remove nontarget molecules from pools containing both. Efficiency of the method was estimated by comparing number of "copies in" to "copies out" by quantitative polymerase chain reaction. PEC retention of DNA targets ranging 109-288 base pairs (bps) in length was 15.88-2.14% (i.e., loss of 84.12-97.86% of target molecules). Experimental modifications of the PEC method resulted in no significant improvements. However, the benefit of PEC was revealed in its ability to remove most nontarget DNA molecules (99.99%). We also discovered that many (56.69%) of the target molecules are "lost" prior to their immobilization on the streptavidin-coated beads. These estimates of methodological efficiency are directly comparable to previous ones observed following "fishing" for DNA, an alternative method for DNA enrichment.

This study sought to document the efficiency of DNA bait capture (i.e., "fishing") methods by two measures: (1) its ability to retain targeted DNA molecules, and (2) its ability to remove non-target DNA molecules from a pool containing both. DNA bait capture uses synthetic biotinylated DNA primers to bind target DNA, which are then immobilized onto streptavidin coated magnetic beads and drawn to a magnet. Bound DNA should, therefore, be isolated from non-target DNA and impurities (e.g., PCR inhibitors) and can be later eluted from the beads for downstream applications. Efficiencies were estimated by comparing the number of "copies in" to "copies out" with quantitative polymerase chain reaction (qPCR). Retention of target DNA molecules, ranging from 109 to 288 base pairs (bps) in length, averaged just 9.06–3.53% (i.e., loss of 90.94–96.47%) using the fishing protocol as originally described. Some improvement was achieved by employing a modified protocol (i.e., with a shortened hybridization time, use of twice the amount of M-270 streptavidin-coated beads, and modified bead washing), resulting in average retention of 31.41–12.08% of the same set of targeted molecules. Noted was the lack of efficacy in removing non-target DNA molecules as opposed to targeted molecules. It was also observed that most of the molecules (61.35–69.49%) are "lost" during the essential hybridization step of the fishing protocol, suggesting its suitability for high copy number samples only. While the bait capture method may be useful in the study of polymerase chain reaction (PCR) inhibited DNA samples as previously suggested, it is necessary to carefully weigh this possible advantage against the degree of expected DNA loss and the non-selectivity of the method for targeted over non-targeted DNA.

DNA from ancient sources is generally believed to be of low copy number, despite minimal attention towards direct measurement of DNA loss accumulated through the extraction process. We developed synthesized “standards” to measure the efficiency of some common DNA extraction methods for degraded skeletal samples, and used quantitative PCR to estimate a known quantity of DNA subjected to a given extraction method (i.e. “copies in”) versus quantity of DNA retained (i.e. “copies out”). All methods performed poorly in retaining short segments of DNA, giving low copy number results even when pre-extraction copy numbers exceeded those expected of ancient samples. These findings challenge low copy number expectations, suggesting that ancient specimens may contain far more preserved genetic material than previously recognized. Furthermore, they emphasize the importance of optimizing and/or developing specialized extraction methods for retrieving degraded DNA.

This study evaluated techniques that can potentially decrease time, cost, and labor in eliminating, circumventing, and/or inactivating as many polymerase chain reaction (PCR) inhibitors as possible while preserving DNA yield. In order to further explore the role of PCR inhibitors in ancient DNA (aDNA) studies, 140 DNA extractions were conducted on 112 Pacific salmonid vertebrae recovered from two archaeological sites located at Dionisio Point on Galiano Island, in southwestern British Columbia, Canada. These DNA extracts and their dilutions at 1:10 and 1:50 were screened for the presence of a 189 base pair (bp) portion of the 12S mitochondrial gene that is used for species identification of Pacific salmonids and other fish. These extracts and their dilutions were also screened for the presence of PCR inhibitors that can cause negative results for amplification of salmonid mtDNA. Repeat silica extraction was conducted on the full concentration extracts until: (1) they either produced a positive result in the salmonid mtDNA reaction, or (2) were deemed to be free of inhibition, but failed to amplify in the salmonid mtDNA reaction. In the latter case, the samples were concluded to not contain sufficient salmonid mtDNA to permit PCR amplification. In obtaining positive salmonid species identification, repeat silica purified extracts (81/133 successes) statistically outperformed dilutions at 1:10 (55/140, p ¼0.0018), 1:50 (63/140, p ¼0.0312), as well as across all dilutions combined (118/280, p ¼0.0025). We also explored the efficacy of EGTA as a decalcifying agent as compared to EDTA, which is commonly employed in aDNA studies. The only extracts that amplified at full concentration (6/140, 4.3%) were those in which EGTA decalcification was used. However, when diluted there was no statistical difference in the success of obtaining positive species identification from bone decalcified with EGTA or EDTA (1:10 and 1:50 dilutions, p ¼0.7891 and p > 0.9999 respectively). From the results of this study, we recommend that aDNA researchers employ greater flexibility in their methodologies as well as be cognizant of the role that PCR inhibitors may play in their investigations.

They Walked Together Paisley Cave in Oregon provides some of the earliest evidence for humans in North America. Jenkins et al. (p. 223 ) provide a wide variety of additional evidence of early human occupation of this site, including a series of radiocarbon ages extending back to nearly 12,500 radiocarbon years ago (about 14,500 calendar years ago). The find includes examples of projectile points representative of the Western Stemmed Tradition dating to about 11,100 radiocarbon years ago. The Western Stemmed Tradition has been thought to have evolved after the dominant Clovis technology, but the find suggests that the two cultures overlapped in time.

Polymerase chain reaction (PCR) inhibitors are often co-extracted with ancient DNA (aDNA) and when present make the analysis of aDNA difficult, if not impossible. In this study we review previous research on PCR inhibitors and techniques that address their co-extraction with DNA from sub-optimal samples. Additionally, we introduce a simple extraction technique, ‘‘repeat silica extraction,’’ that effectively removed PCR inhibitors from extracts of 7000e8000-year-old human skeletal remains from the Windover archaeological site in Florida and 700e2000-yearold human coprolites excavated from Fish Slough Cave in southern California. A series of tests on these same samples demonstrates that N-phenacylthiazolium bromide is largely ineffective, despite previously reported success using this compound as part of the DNA extraction process. We also describe a method for demonstrating the presence as well as successful removal of PCR inhibitors by use of a ‘‘positive aDNA control,’’ a test necessary to conclude that negative PCR amplification is the result of the absence of preserved DNA. 2006 Elsevier Ltd. All rights reserved.

© 2017 Kemp et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The 13thcentury Puebloan depopulation of the Four Corners region of the US Southwest is an iconic episode in world prehistory. Studies of its causes, as well as its consequences, have a bearing not only on archaeological method and theory, but also social responses to climate change, the sociology of social movements, and contemporary patterns of cultural diversity. Previous research has debated the demographic scale, destinations, and impacts of Four Corners migrants. Much of this uncertainty stems from the substantial differences in material culture between the Four Corners vs. hypothesized destination areas. Comparable biological evidence has been difficult to obtain due to the complete departure of farmers from the Four Corners in the 13thcentury CE and restrictions on sampling human remains. As an alternative, patterns of genetic variation among domesticated species were used to address the role of migration in this collapse. We collected mitochondrial haplotypic data from dog (Canis lupus familiaris) and turkey (Meleagris gallopavo) remains from archaeological sites in the most densely-populated portion of the Four Corners region, and the most commonly proposed destination area for that population under migration scenarios. Results are consistent with a large-scale migration of humans, accompanied by their domestic turkeys, during the 13thcentury CE. These results support scenarios that suggest contemporary Pueblo peoples of the Northern Rio Grande are biological and cultural descendants of Four Corners populations.

DNA from ancient and forensic specimens is often co-extracted with unknown amounts of unknown PCR inhibitors, which can lead to underestimated DNA concentrations, allelic drop-out, and/or false-negative results. It is not surprising, in this case, that numerous methods have been developed to remove PCR inhibitors or subdue their effects. One simple and cost effective approach could be the adoption of a polymerase that overcomes or is less affected by PCR inhibitors. In this study, nine different polymerases were evaluated for their efficacy against PCR inhibitors co-extracted with DNA from 63 ancient salmon vertebrae. These samples were excavated from two archeological sites located at the Dionisio Point locality on the northern end of Galiano Island in coastal southwestern British Columbia, Canada and date to 700-1000 and 1300-1500 years before present. Previously, DNA extracts from samples studied from this locality were determined to be largely inhibited to PCR amplification. In the present study, Omni Klentaq LA (DNA Polymerase Technology, Inc.) outperformed the other 8 polymerases in two measures: (1) its success in genetic species identification of these vertebrae, and (2) its ability to amplify an ancient DNA positive control when spiked with a volume of potentially inhibited extract from the vertebrae.

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. To address this issue, a comparative study was designed to examine both cloned and direct sequences amplified from ∼3,500 year-old ancient northern fur seal DNA extracts. Majority rules and the Consensus Confidence Program were used to generate consensus sequences for each individual from the cloned sequences, which exhibited damage at 31 of 139 base pairs across all clones. In no instance did the consensus of clones differ from the direct sequence. This study demonstrates that, when appropriate, cloning need not be the default method, but instead, should be used as a measure of authentication on a case-by-case basis, especially when this practice adds time and cost to studies where it may be superfluous.

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.