DNA release from plant tissue using focused ultrasound extraction (FUSE).

Premise: Sample preparation in genomics is a critical step that is often overlooked in molecular workflows and impacts the success of downstream genetic applications. This study explores the use of a recently developed focused ultrasound extraction (FUSE) technique to enable the rapid release of DNA from plant tissues for genetic analysis. Methods: FUSE generates a dense acoustic cavitation bubble cloud that pulverizes targeted tissue into acellular debris. This technique was applied to leaf samples of American chestnut (Castanea dentata), tulip poplar (Liriodendron tulipifera), red maple (Acer rubrum), and chestnut oak (Quercus montana). Results: We observed that FUSE can extract high quantities of DNA in 9-15 min, compared to the 30 min required for control DNA extraction methods. FUSE extracted DNA quantities of 24.33 ± 6.51 ng/mg and 35.32 ± 9.21 ng/mg from American chestnut and red maple, respectively, while control methods yielded 6.22 ± 0.87 ng/mg and 11.51 ± 1.95 ng/mg, respectively. The quality of the DNA released by FUSE allowed for successful amplification and next-generation sequencing. Discussion: These results indicate that FUSE can improve DNA extraction efficiency for leaf tissues. Continued development of this technology aims to adapt to field-deployable systems to increase the cataloging of genetic biodiversity, particularly in low-resource biodiversity hotspots.

Evaluating the Efficiency of Primer Extension Capture as a Method to Enrich DNA Extractions.

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.

Combating transnational organized crime by linking multiple large ivory seizures to the same dealer.

Rapid growth in world trade has enabled transnational criminal networks to conceal their contraband among the 1 billion containers shipped worldwide annually. Forensic methods are needed to identify the major cartels moving the contraband into transit. We combine DNA-based sample matching and geographic assignment of tusks to show that the two tusks from the same elephant are often shipped by the same trafficker in separate large consignments of ivory. The paired shipments occur close in time from the same initial place of export and have high overlap in the geographic origins of their tusks. Collectively, these paired shipments form a linked chain that reflects the sizes, interconnectedness, and places of operation of Africa's largest ivory smuggling cartels.

Are we fishing or catching? Evaluating the efficiency of bait capture of CODIS fragments.

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.

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.

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.

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.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.