Ancient DNA extraction from bones and teeth

This method is designed to maximize recovery of PCR-amplifiable DNA from ancient bone and teeth specimens and at the same time to minimize co-extraction of substances that inhibit PCR. This is achieved by a combination of DNA extraction from bone powder using a buffer consisting solely of EDTA and proteinase K, and purification of the DNA by binding to silica in the presence of high concentrations of guanidinium thiocyanate. All steps are performed at room temperature (20-23 degrees C), thereby reducing further degradation of the already damaged and fragile ancient DNA and providing an optimal trade-off between DNA release and degradation. Furthermore, the purification step removes most of the various types of PCR inhibitors present in ancient bone samples, thereby optimizing the amount of ancient DNA available for subsequent enzymatic manipulation, such as PCR amplification. The protocol presented here allows DNA extraction from ancient bone and teeth with a minimum of working steps and equipment and yields DNA extracts within 2 working days.     

Radiation-dependent limit for the viability of bacterial spores in halite fluid inclusions and on Mars

When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and S. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old (Vreeland et al., Nature 407, 897-900, 2000). B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus (Helgason et al., Appl. Environ. Microbiol. 66, 2627-2630, 2000). To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the uppermost meter of Mars.     

Ancient saltern metagenomics: tracking changes in microbes and their viruses from the underground to the surface

Microbial communities in hypersaline underground waters derive from ancient organisms trapped within the evaporitic salt crystals and are part of the poorly known subterranean biosphere. Here, we characterized the viral and prokaryotic assemblages present in the hypersaline springs that dissolve Triassic-Keuper evaporite rocks and feed the Añana Salt Valley (Araba/Alava, Basque Country, Spain). Four underground water samples (around 23% total salinity) with different levels of exposure to the open air were analysed by means of microscopy and metagenomics. Cells and viruses in the spring water had lower concentrations than what are normally found in hypersaline environments and seemed to be mostly inactive. Upon exposure to the open air, there was an increase in activity of both cells and viruses as well as a selection of phylotypes. The underground water was inhabited by a rich community harbouring a diverse set of genes coding for retinal binding proteins. A total of 35 viral contigs from 15 to 104 kb, representing partial or total viral genomes, were assembled and their evolutionary changes through the spring system were followed by SNP analysis and metagenomic island tracking. Overall, both the viral and the prokaryotic assemblages changed quickly upon exposure to the open air conditions.     

Ancient DNA from Ascaris: extraction amplification and sequences from eggs collected in coprolites.

On the Middle-Age site of Namur (Belgium) the analysis of coprolites revealed the presence of many well-preserved Ascaris eggs. Following rehydratation of the coprolite samples, 104 eggs were collected and extracted with an ultrasonication and phenol-chloroform based method. Three overlapping fragments of the 18S rRNA gene and one fragment of the cytochrome b gene have been reproducibly amplified, cloned and sequenced. The analysis of these sequences confirms the identification of the eggs as coming from Ascaris. Our study reveals that coprolites can be an interesting source of parasites that can be readily identified using molecular approaches. The study of ancient DNA from helminth parasites is of interest as it may answer long-standing questions in the history of infectious diseases and gives a possibility to compare these ancient sequences with those of modern populations.     

An optimized method for the extraction of ancient eukaryote DNA from marine sediments

Marine sedimentary ancient DNA (sedaDNA) provides a powerful means to reconstruct marine palaeo‐communities across the food web. However, currently there are few optimized sedaDNA extraction protocols available to maximize the yield of small DNA fragments typical of ancient DNA (aDNA) across a broad diversity of eukaryotes. We compared seven combinations of sedaDNA extraction treatments and sequencing library preparations using marine sediments collected at a water depth of 104 m off Maria Island, Tasmania, in 2018. These seven methods contrasted frozen versus refrigerated sediment, bead‐beating induced cell lysis versus ethylenediaminetetraacetic acid (EDTA) incubation, DNA binding in silica spin columns versus in silica‐solution, diluted versus undiluted DNA in shotgun library preparations to test potential inhibition issues during amplification steps, and size‐selection of low molecular‐weight (LMW) DNA to increase the extraction efficiency of sedaDNA. Maximum efficiency was obtained from frozen sediments subjected to a combination of EDTA incubation and bead‐beating, DNA binding in silica‐solution, and undiluted DNA in shotgun libraries, across 45 marine eukaryotic taxa. We present an optimized extraction protocol integrating these steps, with an optional post‐library LMW size‐selection step to retain DNA fragments of ≤500 base pairs. We also describe a stringent bioinformatic filtering approach for metagenomic data and provide a comprehensive list of contaminants as a reference for future sedaDNA studies. The new extraction and data‐processing protocol should improve quantitative paleo‐monitoring of eukaryotes from marine sediments, as well as other studies relying on the detection of highly fragmented and degraded eukaryote DNA in sediments.     

An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis

An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze–thaw–SDS–Protein K) and FTSPP (Freeze–thaw–SDS–Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 μg μL^?1 BSA. However, the FTSPP extraction method with DNA purification by a Wizard^® kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost.     

响应面法优化微波辅助提取发酵虫草菌丝体多糖工艺

为优化发酵虫草菌粉多糖的微波辅助提取工艺,在单因素实验基础上,以液固比、微波功率以及提取时间为自变量,多糖提取率为响应值,采用中心组合设计的方法,研究各自变量及其交互作用对多糖提取率的影响。利用SAS软件和响应面分析相结合的方法对发酵虫草菌粉多糖的微波辅助提取工艺进行优化,确定了微波辅助提取多糖的最佳条件：液固比值12．2,微波功率650．5W,提取时间11．8min,在此条件下,多糖提取率达到6．41％。采用此法提取的虫草菌丝体多糖,当质量浓度为1mg／mL时,对二苯代苦味肼基自由基（DPPH）清除率达到76％。     

Ultrasound-assisted aqueous extraction of total flavonoids and hydroxytyrosol from olive leaves optimized by response surface methodology

Abstract

Olive leaves were often extracted with methanol or ethanol at different proportions. In this study, ultrasound-assisted aqueous extraction was adopted for olive leaf extraction. The yields of total flavonoids (TF) and hydroxytyrosol (HT) were optimized by central composite experimental design. Two second-order polynomial equations were established to quantify the relationship between the responses and the processing parameters. Under the optimal condition of extracting at 60?°C for 60?min with the solvent-to-material ratio of 40, TF and HT amounted to 57.31?±?0.35 and 1.80?±?0.02?mg/g dry leaves (DL), respectively. The scavenging rate of all extracts against α, α-diphenyl-β-picrylhydrazyl (DPPH) and hydroxyl free radicals was screened. The integrated scores, representing both active ingredients and antioxidant capacity of the extracts, were calculated by principle component analysis (PCA). The optimal extract gained the highest score in PCA. In addition, compared to the extracts from 80% methanol to 44% ethanol, the ultrasound-assisted aqueous extract was richer in TF, HT, and polyphenols, while it also presented stronger ferric reducing antioxidant power (FRAP), but poorer strength to quench hydroxyl radicals. The study indicated that the aqueous extract of olive leaves may present broad potential opportunities in health-care sector.     

Spherical particles of halophilic archaea correlate with exposure to low water activity - implications for microbial survival in fluid inclusions of ancient halite

Viable extremely halophilic archaea (haloarchaea) have been isolated from million‐year‐old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000‐ to 34 000‐year‐old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440–454). Searching for a method to produce such particles in the laboratory, we exposed rod‐shaped cells of Halobacterium species to reduced external water activity (a_w). Gradual formation of spheres of about 0.4 μm diameter occurred in 4 m NaCl buffer of a_w ≤ 0.75, but exposure to buffered 4 m LiCl (a_w ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re‐grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50‐fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering a_w should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.     

Experiments in DNA extraction and PCR amplification from bighorn sheep feces: the importance of DNA extraction method

Reliability of genotyping is an issue for studies using non-invasive sources of DNA. We emphasize the importance of refining DNA extraction methods to maximize reliability and efficiency of genotyping for such DNA sources. We present a simple and general method to quantitatively compare genotyping reliability of various DNA extraction techniques and sample materials used. For bighorn sheep (Ovis canadensis) fecal samples we compare different fecal pellet materials, different amounts of fecal pellet material, and the effects of eliminating two DNA extraction steps for four microsatellite loci and four samples heterozygous at each locus. We evaluated 192 PCR outcomes for each treatment using indices of PCR success and peak height (signal strength) developed from analysis output of sequencer chromatograms. Outermost pellet material produced PCR results almost equivalent to DNA extracted from blood. Where any inner pellet material was used for DNA extraction, PCR results were poorer and inconsistent among samples. PCR success was not sensitive to amount of pellet material used until it was decreased to 15 mg from 60 mg. Our PCR index provides considerably more information relative to potential genotyping errors than simply comparing genotypes derived from paired fecal and blood or tissue samples. Our DNA extraction method probably has wide applicability to herbivores that produce pelleted feces where samples dry rapidly after deposition.     

DNA extraction from crayfish exoskeleton

Crayfish exoskeleton (CE) samples are generally less invasive and easy to be collected. However, it is difficult to extract DNA from them. This study was intended to investigate CE as a DNA source and design an easy and efficient DNA extraction protocol for polymerase chain reactions. Specific primer pair (PPO-F, PPO-R) was used to amplify extracted DNA from CE, and compared to crayfish tail muscle DNA sample. Moreover, seven microsatellites markers were used to amplify the CE DNA samples set. Since the extracted DNA from CE is suitable for gene amplification, the results present usefulness of CE as an easy and convenient DNA source for PCR-based population genetic research.     

Ancient DNA from a pre-Columbian Amerindian population

Ancient DNA was obtained from skeletal remains from the Norris Farms #36 cemetery, a pre-Columbian archeological site in central Illinois that dates to A. D. 1300. Four mitochondrial DNA (mtDNA) markers were analyzed that delineate the four primary mtDNA lineages found in contemporary Amerindian populations. mtDNA types were determined for 50 individuals; 49 belonged to one of these four lineages. One lineage occurred only in males, suggesting an immigration of maternally related males into this community. There was no significant spatial patterning of mtDNA lineages within the cemetery. This survey of ancient DNA variation in a preColumbian population supports the view that the initial colonization of the New World comprised just four primary mtDNA lineages. © 1993 Wiley-Liss, Inc.