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Daqu is a typical multi-enzyme and multi-microbes fermentation starter of traditional Chinese liquor. The culture-independent molecular method has become a general method for the study of the Daqu microbial community. To determine the most suitable DNA extraction method of Daqu samples, the effects of different DNA extraction methods on Daqu genomic DNA extraction and amplicon libraries were evaluated. In this study, four DNA extraction methods were selected for the extraction of microbial DNA from two kinds of Daqu. Combined with DNA extraction quality, qPCR quantitative results, Illumina Miseq sequencing results of bacterial 16S rRNA gene, and fungal ITS gene amplicon library, the effects of different extraction methods of Daqu genomic DNA were evaluated. The comprehensive analysis of DNA yield and microbial biomass showed that the DNA yield of the SDS-based extraction method was higher than that of the kit extraction method, and the DNA yield of the in-situ SDS-based extraction method was as high as 5.46 x 104 ng/g, but the DNA purity was low. The qPCR quantitative results of the SDS-based extraction method were 101 - 6 orders of magnitude higher than those of the kit extraction method, and the microbial biomass of the washing-pretreatment plus SDS-based extraction method was the highest, which was 101-3 orders of magnitude higher than that of the in-situ SDS-based extraction method. Whereas the microbial biomass of the DNA extraction kit was the lowest, with only 102-4of the Shannon diversity index among amplicon libraries showed that different DNA extraction methods had significant differences among fungal ITS amplicon libraries, but no significant differences among bacterial 16S rDNA amplicon libraries. The Shannon index of the fungal amplicon library of the washing-pretreated samples were higher than that of the in-situ treated samples. By comparing the operational taxonomic units (OTU) with significant differences among the amplicon libraries, it could be seen that most of the OTUs in the bacterial 16S rDNA amplicon library had differences in abundance, but most of them could be detected in different DNA extraction methods. However, among the fungal ITS amplicon libraries, most OTUs showed a similar trend between different DNA extraction methods, indicating that the DNA extraction method had a significant effect on the extraction of some fungal microbial genomes, and the washing-pretreatment was beneficial to the extraction of fungal genomic DNA. Compared with the four DNA extraction methods, the washing-pretreatment plus SDS-based extraction method was the most suitable for the quantitative and community structure analysis of Daqu microorganisms. [ABSTRACT FROM AUTHOR]

Abstract Microbes from oil reservoirs shape petroleum composition through processes such as biodegradation or souring. Such processes are considered economically detrimental and might pose health and safety hazards. It is therefore crucial to understand the composition of a reservoir's microbial community and its metabolic capabilities. However, such analyses are hindered by difficulties in extracting DNA from such complex fluids as crude oil. Here, we present a novel DNA extraction method from oils with a wide American Petroleum Institute (API) gravity (density) range. We investigated the ability to extract cells from oils with different solvents and surfactants, the latter both nonionic and ionic. Furthermore, we evaluated three DNA extraction methods. Overall, the best DNA yields and the highest number of 16S rRNA reads were achieved with isooctane as a solvent, followed by an ionic surfactant treatment using sodium dodecyl sulfate and DNA extraction using the PowerSoil Pro Kit (Qiagen). The final method was then applied to various oils from oil reservoirs collected in aseptic conditions. Despite the expected low cell density of 101–103 cells/ml, the new method yielded reliable results, with average 16S rRNA sequencing reads in the order of 41431 (±8860) per sample. Thermophilic, halophilic, and anaerobic taxa, which are most likely to be indigenous to the oil reservoir, were found in all samples. API gravity and DNA yield, despite the sufficient DNA obtained, did not show a correlation.

Abstract Background Although wild ungulate populations are heavily monitored throughout Europe, we understand little of how parasites affect population dynamics, and there is no systematic, long-term monitoring of parasite diversity and parasite loads. Such monitoring is in part hampered by a lack of time- and cost-effective assay methodologies with high sensitivity and good taxonomic resolution. DNA metabarcoding has been successfully used to characterize the parasitic nemabiome with high taxonomic resolution in a variety of wild and domestic hosts. However, in order to implement this technique in large-scale, potentially non-invasive monitoring of gastrointestinal parasitic nematodes (GIN), protocol optimization is required to maximize biodiversity detection, whilst maintaining time- and cost-effectiveness. Methods Faecal samples were collected from a wild moose population and GIN communities were characterized and quantified using both parasitological techniques (egg and larva counting) and DNA metabarcoding of the ITS2 region of rDNA. Three different isolation methods were compared that differed in the volume of starting material and cell lysis method. Results Similar nematode faunas were recovered from all samples using both parasitological and metabarcoding methods, and the approaches were largely congruent. However, metabarcoding assays showed better taxonomic resolution and slightly higher sensitivity than egg and larvae counts. The metabarcoding was not strictly quantitative, but the proportion of target nematode sequences recovered was correlated with the parasitologically determined parasite load. Species detection rates in the metabarcoding assays were maximized using a DNA isolation method that included mechanical cell disruption and maximized the starting material volume. Conclusions DNA metabarcoding is a promising technique for the non-invasive, large-scale monitoring of parasitic GINs in wild ungulate populations, owing to its high taxonomic resolution, increased assay sensitivity, and time- and cost-effectiveness. Although metabarcoding is not a strictly quantitative method, it may nonetheless be possible to create a management- and conservation-relevant index for the host parasite load from this data. To optimize the detection rates and time- and cost-effectiveness of metabarcoding assays, we recommend choosing a DNA isolation method that involves mechanical cell disruption and maximizes the starting material volume. Graphical Abstract

Background: Although wild ungulate populations are heavily monitored throughout Europe, we understand little of how parasites affect population dynamics, and there is no systematic, long-term monitoring of parasite diversity and parasite loads. Such monitoring is in part hampered by a lack of time- and cost-effective assay methodologies with high sensitivity and good taxonomic resolution. DNA metabarcoding has been successfully used to characterize the parasitic nemabiome with high taxonomic resolution in a variety of wild and domestic hosts. However, in order to implement this technique in large-scale, potentially non-invasive monitoring of gastrointestinal parasitic nematodes (GIN), protocol optimization is required to maximize biodiversity detection, whilst maintaining time- and cost-effectiveness. Methods: Faecal samples were collected from a wild moose population and GIN communities were characterized and quantified using both parasitological techniques (egg and larva counting) and DNA metabarcoding of the ITS2 region of rDNA. Three different isolation methods were compared that differed in the volume of starting material and cell lysis method. Results: Similar nematode faunas were recovered from all samples using both parasitological and metabarcoding methods, and the approaches were largely congruent. However, metabarcoding assays showed better taxonomic resolution and slightly higher sensitivity than egg and larvae counts. The metabarcoding was not strictly quantitative, but the proportion of target nematode sequences recovered was correlated with the parasitologically determined parasite load. Species detection rates in the metabarcoding assays were maximized using a DNA isolation method that included mechanical cell disruption and maximized the starting material volume. Conclusions: DNA metabarcoding is a promising technique for the non-invasive, large-scale monitoring of parasitic GINs in wild ungulate populations, owing to its high taxonomic resolution, increased assay sensitivity, and time- and cost-effectiveness. Although metabarcoding is not a strictly quantitative method, it may nonetheless be possible to create a management- and conservation-relevant index for the host parasite load from this data. To optimize the detection rates and time- and cost-effectiveness of metabarcoding assays, we recommend choosing a DNA isolation method that involves mechanical cell disruption and maximizes the starting material volume. [ABSTRACT FROM AUTHOR]

In order to establish the general method for fast extracting the colony DNA from Staphylococcus aureus for PCR amplification, and compare the effects of different extraction methods on the extraction of colony DNA, the colonies of Staphylococcus aureus were treated by the boiling method, freeze-thaw method and microwave method, and the DNA was extracted. Then, through observing the effect of PCR amplification, the shortest time for obtaining DNA by the three methods and the concentration of DNA extracted after 10 s of treatment were compared. The results showed that all the three methods could successfully extract the colony DNA from Staphylococcus aureus, and were universal to the common Staphylococcus aureus. These three methods could extract the colony DNA in only 10 s, without time dependence. After 10 s of treatment, the concentration of DNA extracted by the boiling method was significantly higher than that by the freeze-thaw method and microwave method. In summary, the boiling method, freeze-thaw method and microwave method all had better results for the extraction of colony DNA from the common Staphylococcus aureus. By comparison, the boiling method was more recommended for the extraction of colony DNA. [ABSTRACT FROM AUTHOR]

DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water. HIGHLIGHTS DNA extraction methods were evaluated to quantify H. pylori in water.; The tissue kit was suitable for DNA extraction from treated drinking water.; The stool kit was efficient in analyzing samples from the river after heavy rain.; The soil kit could extract DNA effectively from the river and stream with no rain.; The characteristics of the water sample were important in detecting H. pylori.;

Abstract Background As an important player during food digestion, gut microbiota has attracted much attention in diet adaptation studies in birds. Microbiota extracted from feces has been widely used as a proxy for gut microbiota. Although several methods have been developed for microbial DNA extraction, their performances in the bird feces have not been systematacially evaluated yet. Methods In this study, we applied three DNA extraction methods (Qiagen, MoBio and Bead) to extract DNA from feces of three avian dietary guilds (granivore, omnivore and carnivore), sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield, DNA integrity, microbial composition, cell lysis capacity and alpha diversity for the three methods on each dietary guild. Results Bead method was the best on the performance of both DNA yield and DNA integrity regardless of dietary guild. In granivore, microbial relative abundance at both species and phylum levels, alpha diversity and cell lysis capacity were comparable among all methods. In omnivore, Qiagen had the best performance on alpha diversity, followed by Bead and MoBio. There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods. MoBio exhibited the best performance on cell lysis capacity. In carnivore, considerable variations were found on microbial relative abundance at both species and phylum levels. Qiagen had the best performance on alpha diversity, followed by MoBio and Bead. MoBio had the highest cell lysis capacity. Conclusions DNA yield and integrity have no obvious impact on microbial composition, alpha diversity or cell lysis capacity. The microbiota results (e.g., microbial composition, cell lysis capacity, alpha diversity) obtained from different methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds. Either method could be used in granivore microbiota studies. For omnivores and carnivores, we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target.

Background and Objectives: DNA extraction is an important step of any molecular experiment. DNA could not be easily extracted from members of actinomycetes by the usual methods of lysis. Due to the low efficiency of the conventional DNA extraction methods, development of an effective technique for DNA extraction of actinobacteria in emergency cases seems to be necessary. Since most of the DNA extraction techniques and commercial kits are not efficient enough to extract DNA from actinobacteria, this study was conducted to improve an efficient method obtained from conventional one to extract DNA from this group of bacteria. Materials and Methods: DNA extraction was performed using five methods (an improved method, Invisorb Spin Plant Mini Kit, EZ-10 Spin Column, Sarrbrucken method (HZI, Germany) and Kirby Bauer's method). To evaluate the quantity and quality of extracted genomic DNA, UV absorbance of all samples and efficiency of polymerase chain reaction (PCR) were evaluated. Results: Overall, the results revealed that the highest quantity (up to 4000 ng/µl) and good quality of DNA was obtained using introduced DNA extraction method. Conclusion: Results indicated that recently introduced improved method was more efficient for extraction of DNA from actinobacteria for DDH (DNA-DNA hybridization) test and for those require the high concentration of DNA. [ABSTRACT FROM AUTHOR]

Background and Objectives: DNA extraction is an important step of any molecular experiment. DNA could not be easily extracted from members of actinomycetes by the usual methods of lysis. Due to the low efficiency of the conventional DNA extraction methods, development of an effective technique for DNA extraction of actinobacteria in emergency cases seems to be necessary. Since most of the DNA extraction techniques and commercial kits are not efficient enough to extract DNA from actinobacteria, this study was conducted to improve an efficient method obtained from conventional one to extract DNA from this group of bacteria. Materials and Methods: DNA extraction was performed using five methods (an improved method, Invisorb Spin Plant Mini Kit, EZ-10 Spin Column, Sarrbrucken method (HZI, Germany) and Kirby Bauer's method). To evaluate the quantity and quality of extracted genomic DNA, UV absorbance of all samples and efficiency of polymerase chain reaction (PCR) were evaluated. Results: Overall, the results revealed that the highest quantity (up to 4000 ng/µl) and good quality of DNA was obtained using introduced DNA extraction method. Conclusion: Results indicated that recently introduced improved method was more efficient for extraction of DNA from actinobacteria for DDH (DNA–DNA hybridization) test and for those require the high concentration of DNA.

With the advent of new molecular diagnostic techniques, retrieving DNA from the formalin-fixed paraffin-embedded (FFPE) tissues has become an essential yet challenging step for efficient downstream processes. Owing to low quality and quantity of DNA retrieved from the FFPE sections, the process is often impractical and needs significant improvements. Here, we established an efficient method for the purification of DNA from FFPE specimens by optimizing incubation temperature, incubation time, and the concentration of a formalin scavenger tris(hydroxymethyl)aminomethane (Tris) for reverse-crosslinking. The optimized method, named " Hi ghly concentrated T ris-mediated DNA e xtraction" (HiTE), yielded three times the DNA yield per tissue slice compared with a representative DNA extraction kit. Moreover, the use of HiTE-extracted DNA increased the yield of the sequencing library three times and accordingly yielded a log higher and more reproducible sequencing library compared with that obtained using the commonly used commercial kit. The sequencing library prepared from HiTE-extracted FFPE-DNA had longer inserts and produced reads that evenly covered the reference genome. Successful application of HiTE-extracted FFPE-DNA for whole-genome and targeted gene panel sequencing indicates its practical usability. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Although protocols exist for the recovery of ancient DNA from land snail and marine bivalve shells, marine conch shells have yet to be studied from a paleogenomic perspective. We first present reference assemblies for both a 623.7 Mbp nuclear genome and a 15.4 kbp mitochondrial genome for Strombus pugilis, the West Indian fighting conch. We next detail a method to extract and sequence DNA from conch shells and apply it to conch from Bocas del Toro, Panama across three time periods: recently‐eaten and discarded (n = 3), Late Holocene (984–1258 before present [BP]) archaeological midden (n = 5), and mid‐Holocene (5711–7187 BP) paleontological fossil coral reef (n = 5). These results are compared to control DNA extracted from live‐caught tissue and fresh shells (n = 5). Using high‐throughput sequencing, we were able to obtain S. pugilis nuclear sequence reads from shells across all age periods: up to 92.5 thousand filtered reads per sample in live‐caught shell material, 4.57 thousand for modern discarded shells, 12.1 thousand reads for archaeological shells, and 114 reads in paleontological shells. We confirmed authenticity of the ancient DNA recovered from the archaeological and paleontological shells based on 5.7× higher average frequency of deamination‐driven misincorporations and 15% shorter average read lengths compared to the modern shells. Reads also mapped to the S. pugilis mitochondrial genome for all but the paleontological shells, with consistent ratios of mitochondrial to nuclear mapped reads across sample types. Our methods can be applied to diverse archaeological sites to facilitate reconstructions of the long‐term impacts of human behaviour on mollusc evolutionary biology. [ABSTRACT FROM AUTHOR]

[Display omitted] • 305 fish samples covering six types of processed products were evaluated. • SALT method was determined as the most appropriate DNA extraction method. • Approximately 36.4% of fish samples were inconsistent with the labels. • Mini-barcoding can precisely identify processed products containing degraded DNA. DNA-based methods are reliable for a precise identification of species in processed products. In this study, we assessed five typical DNA extraction methods from multiple aspects. Full-length and mini-length DNA barcoding were performed to detect the species substitution and mislabeling of 305 processed fish products from the Chinese market covering six processed fish products. The salt extraction method that exhibited the best overall performance was applied. All samples were successfully extracted; however, only 19.3 % of samples could be amplified using the full-DNA barcode primer set, and 90.2 % of samples could be amplified using the newly designed mini-DNA barcode primer sets (401 and 320 bp). Overall, the molecular identification results revealed that 36.4 % (111/305) of the samples were inconsistent with the labels, with commercial fraud observed in all six types of processed fish products. The survey findings provide technical references for effective fish authentication monitoring, offering insights into the seafood safety in markets. [ABSTRACT FROM AUTHOR]

Background: As an important player during food digestion, gut microbiota has attracted much attention in diet adaptation studies in birds. Microbiota extracted from feces has been widely used as a proxy for gut microbiota. Although several methods have been developed for microbial DNA extraction, their performances in the bird feces have not been systematacially evaluated yet. Methods: In this study, we applied three DNA extraction methods (Qiagen, MoBio and Bead) to extract DNA from feces of three avian dietary guilds (granivore, omnivore and carnivore), sequenced V4 region of 16S rRNA gene for each extract and evaluated the performances of DNA yield, DNA integrity, microbial composition, cell lysis capacity and alpha diversity for the three methods on each dietary guild. Results: Bead method was the best on the performance of both DNA yield and DNA integrity regardless of dietary guild. In granivore, microbial relative abundance at both species and phylum levels, alpha diversity and cell lysis capacity were comparable among all methods. In omnivore, Qiagen had the best performance on alpha diversity, followed by Bead and MoBio. There were small variations on microbial relative abundance at both species and phylum levels among different extraction methods. MoBio exhibited the best performance on cell lysis capacity. In carnivore, considerable variations were found on microbial relative abundance at both species and phylum levels. Qiagen had the best performance on alpha diversity, followed by MoBio and Bead. MoBio had the highest cell lysis capacity. Conclusions: DNA yield and integrity have no obvious impact on microbial composition, alpha diversity or cell lysis capacity. The microbiota results (e.g., microbial composition, cell lysis capacity, alpha diversity) obtained from different methods are comparable in granivorous avian species but not in omnivorous or carnivorous birds. Either method could be used in granivore microbiota studies. For omnivores and carnivores, we recommend Qiagen method when the research purpose is microbial diversity and MoBio when gram-positive bacteria is the research target. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Henan University of Technology Natural Science Edition is the property of Henan University of Technology Journal Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In this study, strain Streptomyces sp. Act4Zk was isolated based on a method developed for the isolation of myxobacteria. Due to the low efficiency of the majority of conventional DNA extraction techniques, for molecular identification of the strain Streptomyces sp. Act4Zk, a new technique for DNA extraction of Actinobacteria was developed. In order to explore potential bioactivities of the strain, extracts of the fermented broth culture were prepared by an organic solvent (i.e. ethyl acetate) extraction method using. These ethyl acetate extracts were subjected to HPLC fractionation against standard micro‐organisms, followed by LC/MS analysis. Based on morphological, physiological, biochemical and 16S rRNA gene sequence data, strain Streptomyces sp. Act4Zk is likely to be a new species of Streptomyces, close to Streptomyces genecies and Streptomyces roseolilacinus. Antimicrobial assay indicated high antifungal activity as well as antibacterial activity against Mycobacterium smegmatis and Gram‐positive bacteria for the new strain. HPLC and LC/MS analyses of the extracts led to the identification of three different compounds and confirmed our hypothesis that the interesting species of the genus Streptomyces being a good producer of staurosporine and some derivatives. [ABSTRACT FROM AUTHOR]

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

Both the host microbiome and the microbiome of the built environment can have profound impacts on human health. While prior studies have suggested that the variability introduced by DNA extraction method is less than typical biologic variation, most studies have focused on 16S rRNA amplicon sequencing or on high biomass fecal samples. Shotgun metagenomic sequencing provides advantages over amplicon sequencing for surveying the microbiome, but is a challenge to perform in lower microbial biomass samples with high human DNA content such as sputum or vacuumed dust. Here we systematically evaluate the impact of four different extraction methods (phenol:choloroform, and three high-throughput kit-based approaches, the Promega Maxwell gDNA, Qiagen MagAttract PowerSoil DNA, and ZymoBIOMICS 96 MagBead). We report the variation in microbial community structure and predicted microbial function assessed by shotgun metagenomics sequencing in human stool, sputum, and vacuumed dust obtained from ongoing cohort studies or clinical trials. The same beadbeating protocol was used for all samples to focus our evaluation on the impact of kit chemistries on sequencing results. DNA yield was overall highest in the phenol:choloroform and Promega approaches. Only the phenol:choloroform approach showed evidence of contamination in negative controls. Bias was evaluated using mock community controls, and was noted across all extraction methods, although Promega exhibited the least amount of bias. The extraction method did not impact the proportion of human reads, although stool had the lowest proportion of human reads (0.1%) as compared to dust (44.1%) and sputum (80%). We calculated Bray-Curtis dissimilarity and Aitchison distances to evaluate the impact of extraction method on microbial community structure by sample type. Extraction method had the lowest impact in stool (extraction method responsible for 3.0–3.9% of the variability), the most impact in vacuumed dust (12–16% of the variability) and intermediate values for sputum (9.2–12% variability). Similar differences were noted when evaluating microbial community function. Our results will inform investigators planning microbiome studies using diverse sample types in large clinical studies. A consistent DNA extraction approach across all sample types is recommended, particularly with lower microbial biomass samples that are more heavily influenced by extraction method.

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform. [ABSTRACT FROM AUTHOR]

For identification of badly preserved cadavers, only a few tissues can be used as a source of DNA, mostly bones and teeth, from which sampling and DNA extraction are difficult and time-consuming. In most highly decomposed remains, the nails are preserved. The aim of this study was to evaluate nails as an alternative source of DNA instead of bones and teeth in demanding routine identification cases. An automated extraction method was optimized on nails obtained from 33 cadavers with a post-mortem interval (PMI) up to 5 years. The commercially available EZ1 Investigator Kit (Qiagen) was used for extraction, and the G2 buffer included in the kit was replaced with TNCa buffer, and DTT was added for digestion of 5 mg of nail. The DNA was purified in a Biorobot EZ1 device (Qiagen), quantified using the PowerQuant System (Promega), and STR typing was performed with the NGM kit (TFS). From 0.3 to 270 μg DNA/g of nail was obtained from the samples analyzed, with an average yield of 36 μg DNA/g of nail. Full STR profiles were obtained from all nails except one. The optimized extraction method proved to be fast and highly efficient in the removal of PCR inhibitors, and it yields high amounts of DNA for successful STR typing. Nails were implemented as the primary sample type for obtaining DNA from highly decomposed and partially skeletonized cadavers in routine forensic identification cases in our laboratory. [ABSTRACT FROM AUTHOR]

Both the host microbiome and the microbiome of the built environment can have profound impacts on human health. While prior studies have suggested that the variability introduced by DNA extraction method is less than typical biologic variation, most studies have focused on 16S rRNA amplicon sequencing or on high biomass fecal samples. Shotgun metagenomic sequencing provides advantages over amplicon sequencing for surveying the microbiome, but is a challenge to perform in lower microbial biomass samples with high human DNA content such as sputum or vacuumed dust. Here we systematically evaluate the impact of four different extraction methods (phenol:choloroform, and three high-throughput kit-based approaches, the Promega Maxwell gDNA, Qiagen MagAttract PowerSoil DNA, and ZymoBIOMICS 96 MagBead). We report the variation in microbial community structure and predicted microbial function assessed by shotgun metagenomics sequencing in human stool, sputum, and vacuumed dust obtained from ongoing cohort studies or clinical trials. The same beadbeating protocol was used for all samples to focus our evaluation on the impact of kit chemistries on sequencing results. DNA yield was overall highest in the phenol:choloroform and Promega approaches. Only the phenol:choloroform approach showed evidence of contamination in negative controls. Bias was evaluated using mock community controls, and was noted across all extraction methods, although Promega exhibited the least amount of bias. The extraction method did not impact the proportion of human reads, although stool had the lowest proportion of human reads (0.1%) as compared to dust (44.1%) and sputum (80%). We calculated Bray-Curtis dissimilarity and Aitchison distances to evaluate the impact of extraction method on microbial community structure by sample type. Extraction method had the lowest impact in stool (extraction method responsible for 3.0–3.9% of the variability), the most impact in vacuumed dust (12–16% of the variability) and intermediate values for sputum (9.2–12% variability). Similar differences were noted when evaluating microbial community function. Our results will inform investigators planning microbiome studies using diverse sample types in large clinical studies. A consistent DNA extraction approach across all sample types is recommended, particularly with lower microbial biomass samples that are more heavily influenced by extraction method. [ABSTRACT FROM AUTHOR]

Marine free-living (FL) and plankton-associated prokaryotes (plankton-microbiota) are at the basis of trophic webs and play crucial roles in the transfer and cycling of nutrients, organic matter, and contaminants. Different ecological niches exist along the plankton size fraction gradient. Despite its relevant ecological role, the plankton-microbiota has rarely been investigated with a sufficient level of size-fraction resolution, and it can be challenging to study because of overwhelming eukaryotic DNA. Here we compared the prokaryotic diversity obtained by 16S rRNA gene sequencing from six plankton size fractions (from FL to mesoplankton), through three DNA recovery methods: direct extraction, desorption pretreatment, enrichment post-treatment. The plankton microbiota differed strongly according to the plankton size-fraction and methodological approach. Prokaryotic taxa specific to each size fraction, and methodology used, were identified. Vibrionaceae were over-represented by cell desorption pretreatment, while prokaryotic DNA enrichment had taxon-specific effects, indicating that direct DNA extraction was the most appropriate method. [Display omitted] • The plankton microbiota differed strongly according to the plankton size-fraction. • The bulk community (> 0.2 μm) was more similar to the 0.2–90 μm fraction. • Cell desorption pretreatment led to a biased over-representation of Vibrionaceae. • The influence of prokaryotic DNA enrichment was taxon-specific. [ABSTRACT FROM AUTHOR]

Our understanding of human gut microbiota in health and disease depends on accurate and reproducible microbial data acquisition. The critical step in this process is to apply an appropriate methodology to extract microbial DNA, since biases introduced during the DNA extraction process may result in inaccurate microbial representation. In this study, we attempted to find a DNA extraction protocol which could be effectively used to analyze both the bacterial and fungal community. We evaluated the effect of five DNA extraction methods (QIAamp DNA Stool Mini Kit, PureLinkTM Microbiome DNA Purification Kit, ZR Fecal DNA MiniPrepTM Kit, NucleoSpin® DNA Stool Kit, and IHMS protocol Q) on bacterial and fungal gut microbiome recovery using (i) a defined system of germ-free mice feces spiked with bacterial or fungal strains, and (ii) non-spiked human feces. In our experimental setup, we confirmed that the examined methods significantly differed in efficiency and quality, which affected the identified stool microbiome composition. In addition, our results indicated that fungal DNA extraction might be prone to be affected by reagent/kit contamination, and thus an appropriate blank control should be included in mycobiome research. Overall, standardized IHMS protocol Q, recommended by the International Human Microbiome Consortium, performed the best when considering all the parameters analyzed, and thus could be applied not only in bacterial, but also in fungal microbiome research.

Our understanding of human gut microbiota in health and disease depends on accurate and reproducible microbial data acquisition. The critical step in this process is to apply an appropriate methodology to extract microbial DNA, since biases introduced during the DNA extraction process may result in inaccurate microbial representation. In this study, we attempted to find a DNA extraction protocol which could be effectively used to analyze both the bacterial and fungal community. We evaluated the effect of five DNA extraction methods (QIAamp DNA Stool Mini Kit, PureLinkTM Microbiome DNA Purification Kit, ZR Fecal DNA MiniPrepTM Kit, NucleoSpin® DNA Stool Kit, and IHMS protocol Q) on bacterial and fungal gut microbiome recovery using (i) a defined system of germ-free mice feces spiked with bacterial or fungal strains, and (ii) non-spiked human feces. In our experimental setup, we confirmed that the examined methods significantly differed in efficiency and quality, which affected the identified stool microbiome composition. In addition, our results indicated that fungal DNA extraction might be prone to be affected by reagent/kit contamination, and thus an appropriate blank control should be included in mycobiome research. Overall, standardized IHMS protocol Q, recommended by the International Human Microbiome Consortium, performed the best when considering all the parameters analyzed, and thus could be applied not only in bacterial, but also in fungal microbiome research. [ABSTRACT FROM AUTHOR]

Highlights • Maternal STRs were detected in degraded placenta from an abandoned newborn male baby. • Samples collected from the intervillous space maternal blood pool were analyzed. • DNA was extracted at multiple time points and severely degraded cells were lysed. • Two additional lysis steps increased maternal DNA recovery and eliminated fetal DNA. • A good-quality mixed STR profile was obtained by routine forensic methods. Abstract The placenta is a unique and complex organ composed of a mixture of fetal and maternal tissues. In this study, we aimed to detect maternal short tandem repeats (STRs) in degraded placenta from a newborn male baby found abandoned in a river. In order to deduce maternal alleles—which was not possible by sampling of different parts of the placenta—we collected samples from the maternal blood pool in the intervillous space and applied a multi-step method (named tempo-gap DNA extraction) for extracting DNA at defined time points after cell lysis (10 min, 2 h 10 min, and 4 h 10 min). The first lysis step (10 min) effectively removed severely degraded DNA; this was followed by a second lysis step (2 h 10 min) for high recovery of both fetal and maternal DNA. The third lysis step (4 h 10 min) effectively eliminated unwanted residual fetal DNA. The differential lysis of fetal and maternal cells occurred not because fetal and maternal cells exhibited different lysis behavior, but because of the difference in their numbers. Although all of the lysates showed fetal cell contamination, we were able to derive a maternal STR profile from the good-quality mixed STR profile from the second lysate of placental piece B. This study provides technical insight into concurrent issues encountered during routine forensic analysis of DNA samples, such as degradation, cell contamination (mixed DNA), and low-template DNA. [ABSTRACT FROM AUTHOR]

Understanding in population structure of a plant’s root-associated microbiome is applied in good practices in agricultural activities to improve production yields and enhance plant immune responses. The molecular analysis of bacterial populations inhabited in soil faces difficulties in obtaining high yield and high purity of DNA, and different commercial DNA extraction kits have been developed for this purpose. This study focuses on the comparison of DNA extraction of six different rice root-associated bacterial consortium using three commercial kits with two key technologies, spin-column adsorption and magnetic bead adsorption. The quality and quantity of genomic DNA obtained from these extraction methods were analyzed and compared based on DNA concentration, DNA purity and efficiency to be used as a template for 16sRNA amplification. The results showed that the extraction kit with magnetic bead adsorption technology showed the highest concentration (101.32 ng/μl) compared to other DNA extraction kits (32.67 and 1.89 ng/μl). The purity values (A260/A280) were assessed by using Nano-drop spectrophotometer and resulted in purities of nucleic acids in the range of 1.4-1.7. Thus, it was concluded that the extracted DNA obtained from the extraction kit with magnetic bead adsorption technology can be valuable for molecular analysis of microbial communities present in the soil.

Environmental waters (EW) substantially lend to the transmission of Helicobacter pylori (Hp). But the increase in Hp infections and antimicrobial resistance is often attributed to socioeconomic status. The connection between socioeconomic status and Hp prevalence in EW is however yet to be investigated. This study aimed to assess the impacts of socioeconomic indices (SI: continent, world bank region (WBR), world bank income (WBI), WHO region, Socio-demographic Index (SDI quintile), Sustainable Development Index (SuDI), and Human Development Index (HDI)) on the prevalence of Hp in EW. Hp-EW data were fitted to a generalized linear mixed-effects model and SI-guided meta-regression models with a 1000-resampling test. The worldwide prevalence of Hp in EW was 21.76% [95% confidence interval [CI]: 10.29–40.29], which declined significantly from 59.52% [43.28–74.37] in 1990–99 to 19.36% [3.99–58.09] in 2010–19 and with increasing trend in 2020–22 (33.33%, 22.66–45.43). Hp prevalence in EW was highest in North America (45.12%, 17.07–76.66), then Europe (22.38%, 5.96–56.74), South America (22.09%, 13.76–33.49), Asia (2.98%, 0.02–85.17), and Africa (2.56%, 0.00–99.99). It was negligibly different among sampling settings, WBI, and WHO regions demonstrating highest prevalence in rural location [42.62%, 3.07–94.56], HIEs [32.82%, 13.19–61.10], and AMR [39.43%, 19.92–63.01], respectively. However, HDI, sample size, and microbiological method robustly predict Hp prevalence in EW justifying 26.08%, 21.15%, and 16.44% of the true difference, respectively. In conclusion, Hp is highly prevalence in EW across regional/socioeconomic strata and thus challenged the uses of socioeconomic status as surrogate for hygienic/sanitary practices in estimating Hp infection prevalence. [Display omitted] • Environmental waters (EW) add to H. pylori (Hp) spread via (in)direct pathways. • Global Hp prevalence in EW was 21.76% [95%CI: 10.29 – 40.29]. • Human development index, sample size, & methods robustly predict Hp prevalence in EW. • Hp is highly prevalence in EW dismissing regional socioeconomic strength. [ABSTRACT FROM AUTHOR]

Microbes from oil reservoirs shape petroleum composition through processes such as biodegradation or souring. Such processes are considered economically detrimental and might pose health and safety hazards. It is therefore crucial to understand the composition of a reservoir's microbial community and its metabolic capabilities. However, such analyses are hindered by difficulties in extracting DNA from such complex fluids as crude oil. Here, we present a novel DNA extraction method from oils with a wide American Petroleum Institute (API) gravity (density) range. We investigated the ability to extract cells from oils with different solvents and surfactants, the latter both nonionic and ionic. Furthermore, we evaluated three DNA extraction methods. Overall, the best DNA yields and the highest number of 16S rRNA reads were achieved with isooctane as a solvent, followed by an ionic surfactant treatment using sodium dodecyl sulfate and DNA extraction using the PowerSoil Pro Kit (Qiagen). The final method was then applied to various oils from oil reservoirs collected in aseptic conditions. Despite the expected low cell density of 10 1 -10 3  cells/ml, the new method yielded reliable results, with average 16S rRNA sequencing reads in the order of 41431 (±8860) per sample. Thermophilic, halophilic, and anaerobic taxa, which are most likely to be indigenous to the oil reservoir, were found in all samples. API gravity and DNA yield, despite the sufficient DNA obtained, did not show a correlation., Competing Interests: The authors declare no conflict of interests., (© 2023 The Authors. mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.)

Extracellular antibiotic resistance genes (exARGs) constitute a significant type of antibiotic resistance genes. However, current exARGs studies are restricted due to the lack of efficient extraction methods for extracellular DNA (exDNA), which are laborious, dependent on extra experimental agents, and prone to interference by coexisting substances. In this study, we have developed an ultra-selective exDNA extraction method based on molecular imprinting technology. The resulting guanine imprinted beads are micron grade spheres with high capacity (384.6 mg/g) and speed (90% adsorption in 10 min) for DNA adsorption at variable lengths, including ultra-short DNA (<100 bp)， without requiring extra agents. The guanine-imprinted beads demonstrated exclusive DNA adsorption capabilities and avoided interference by other competing substances. Furthermore, guanine imprinted beads effectively extracted DNA at ideal recovery efficiencies (81%∼109%) from diverse environments, such as soil, air, river water and vegetable. Higher abundance of exARGs was obtained by using guanine imprinted beads than currently used commercial magnetic beads. In addition to exDNA extraction quality, a higher diversity of exDNA-releasing bacteria was acquired by guanine imprinted beads. This contributes to 1.2-fold increase in ARG-genera (172 v.s. 150) and 3.6-fold increase in ARG-pathogen associations (96 v.s. 27), indicating a greater number of bacterial hosts of ARGs. This study provides a simple and robust method for advanced exDNA studies, such as discovering novel exARGs and identifying hosts of exARGs. [Display omitted] • A selective extracellular DNA (exDNA) extraction method is developed. • Guanine imprinted beads adsorb exDNA with high affinity without extra agents. • Guanine imprinted beads effectively extract exDNA from various environments. • Extracellular resistance genes are efficiently recovered by guanine imprinted beads. • Guanine imprinted beads induce more ARG-genera and ARG-pathogen associations. [ABSTRACT FROM AUTHOR]

Collecting and obtaining sufficient amount of airborne particles for multiple microbial component assessments can be challenging. A passive dust sampling device, the electrostatic dust fall collector (EDC) has been established for assessing airborne exposures including endotoxin and glucans. Recently, with advances in next-generation sequencing techniques, EDCs were used to collect microbial cells for DNA sequencing analysis to promote the study of airborne bacterial and fungal communities. However, low DNA yields have been problematic when employing passive sampling with EDC. To address this challenge, we attempted to increase the efficiency of extraction. We compared DNA extraction efficiency of bacterial components from EDCs captured on filters through filtration using five extraction techniques. By measuring the abundance, diversity and structure of bacterial communities using qPCR and amplicon sequencing targeting 16S rRNA genes, we found that two techniques outperformed the rest. Furthermore, we developed protocols to simultaneously extract both DNA and endotoxin from a single EDC cloth. Our technique promotes a high quality to price ratio and may be employed in large epidemiological studies addressing airborne bacterial exposure where a large number of samples is needed., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Randi J. Bertelsen reports financial support was provided by European Research Council., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

To rapidly obtain high-quality genomic DNA from Chenopodium quinoa Willd, the genomic DAN in different tissues (leaves, stems and roots) of Chenopodium quinoa Willd was extracted by modified CTAB method, SDS method and high-salt low-pH method, respectively. The quality and yield of extracted DNA was determined using agarose gel electrophoresis and UV spectrophotometry. At the same time, the PCR-SSR and SSCP molecular detection was also performed. The results showed that the gel test strips, without obvious decomposition, of all the extraction methods were relatively obvious; the genomic DNA yield extracted by modified CTAB method was highest, followed by that by SDS method, and the genomic DNA extracted by high-salt low-pH method was lowest; the genomic DNA yields extracted by different methods from Chenopodium quinoa Willd leaves were all higher than those from roots and stems; the quality of Chenopodium quinoa Willd genomic DNA extracted by modified CTAB method and high-salt low-pH method was better, and polyphenols, polysaccharides and other impurities were removed more completely. The PCR-SSR and SSCP detection results showed that the genomic DNA extracted by different methods from different tissues of Chenopodium quinoa Willd all could be better amplified, and high-quality strips could be obtained. So the Chenopodium quinoa Willd genomic DNA extracted by the three methods all can be used for subsequent molecular biology research. [ABSTRACT FROM AUTHOR]

Three nuclear DNA markers that diagnostically differentiate mussels within the Mytilus edulis complex (M. edulis, M. trossulus and M. galloprovincialis) are commonly used in taxonomic investigations: Glu5', ITS and EFbis. As a rule, DNA extraction is performed before amplification. It is a time consuming process in the case of traditional methods based on chloroform and phenol extraction or relatively expensive using kits with ready spin columns. Moreover, DNA isolation from larvae is problematic, because of the small amount of tissue available. In this report we describe a simple, fast and inexpensive method of DNA extraction and gene amplification from larvae, spat and adults of the Baltic mussel Mytilus trossulus. The extraction method is adapted from that of Wang et al. (2006) and is based on digestion of tissue or whole animals in STE solution and direct gene amplification. On the basis of the results of routine analyses of mussels carried out in our laboratory we have concluded that the method we propose gives results that are consistent with standard methods, without requiring expensive reagents/equipment and is time saving. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies have reported an association between shorter leukocyte telomere length and schizophrenia (SCZ). The aim of the present study was to replicate this finding in a large sample of SCZ patients (n=539) and population-based controls (n=519). In addition, the possible influence of SCZ severity on telomere length – as measured by age of onset, mode of onset, and course of the disorder – was investigated. Telomere length was negatively associated with age in both patients and controls. This is a consistently reported phenomenon, related to the problem of DNA end-replication. However, in contrast to previous findings, SCZ patients displayed longer telomeres compared to controls (p=0.015). No association was found with any SCZ-severity subphenotype. Interestingly, recent studies have reported associations between longer leukocyte telomere length and both smaller hippocampal volume, and poorer episodic memory performance. Both phenotypes are common in patients with SCZ. Further studies are warranted to investigate whether the present association between SCZ and increased telomere length was driven by such associations, or rather by association with the clinical disease per se or other associated phenotypes, endophenotypes or lifestyle factors. [Copyright &y& Elsevier]

The article discusses a study that evaluated Porcine circovirus-2 (PCV-2) polymerase chain reaction (PCR) data taken after application of different DNA extraction methods to lymph node specimens. It cites the evaluation of 3 manual DNA extraction methods namely the DNeasy tissue kit, a modified in-house silica-based affinity protocol and DNAzol reagent. It indicate that the DNA extraction method affects PCV-2 quantification in lymph node samples by qPCR.

Abstract: Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol. [Copyright &y& Elsevier]

In this study, the development of a duplex PCR for the specific detection of Aspergillus carbonarius-a potent ochratoxin A (OTA) producer-is described. This approach resulted in a very accurate and sensitive method for the detection of low amounts of the mycotoxigenic mould occurring in grapes. In addition, an optimised protocol for the isolation of genomic DNA from a polyphenol-rich fruit like grape was developed. [ABSTRACT FROM AUTHOR]

Owing to the increasing importance of genomic information, obtaining genomic DNA easily from biological specimens has become more and more important. This article proposes an efficient method for obtaining genomic DNA from nail clippings. Nail clippings can be easily obtained, are thermostable and easy to transport, and have low infectivity. The drawback of their use, however, has been the difficulty of extracting genomic material from them. We have overcome this obstacle using the protease solution obtained from Cucumis melo. The keratinolytic activity of the protease solution was 1.78-fold higher than that of proteinase K, which is commonly used to degrade keratin. With the protease solution, three times more DNA was extracted than when proteinase K was used. In order to verify the integrity of the extracted DNA, genotype analysis on 170 subjects was performed by both PCR–RFLP and Real Time PCR. The results of the genotyping showed that the extracted DNA was suitable for genotyping analysis. In conclusion, we have developed an efficient extraction method for using nail clippings as a genome source and a research tool in molecular epidemiology, medical diagnostics, and forensic science. [ABSTRACT FROM AUTHOR]

We developed a method using an internal standard to determine the amount of unprocessed genetically modified organisms (GMO) in foods as GMO weight per weight of total food (w/wtf) using real-time PCR. Food samples were spiked with an internal standard, a ColE1 plasmid, and DNA was extracted from the samples using a silica membrane-type kit and analysed using real-time PCR. The relationship between the GMO amount and the copy number ratio of the transgene to ColE1 in 0.1–5% w/wtf GM soybean powders was found to have a correlation coefficient ( r) of 0.97. GMO was quantified in food samples spiked with GM soybean (final amount 0.5% w/wtf GMO). The average GMO amount ranged from 0.35% to 0.63% w/wtf. The results show that our method should be useful for quantifying unprocessed GMO in foods. We also developed a duplex assay, which is simpler and more accurate than the simplex assay. [ABSTRACT FROM AUTHOR]

Abstract: A simple noninvasive procedure for saliva sample collection and DNA extraction was developed. On average, the amount of human DNA (as measured by a TaqMan-based assay) was about 11.4μg/mL saliva, which is more than can be obtained from other noninvasive samples such as cheek swabs. However, the presence of large amounts of nonhuman DNA (up to 90% of the total extracted DNA) in saliva samples does necessitate DNA quantitation methods that are specific for human DNA. We were able to reliably and accurately type different genetic markers (mDNA sequences, Y-chromosomal single-nucleotide polymorphisms, and autosomal microsatellite loci) from saliva samples stored for up to 30 days at 37°C, making this method well-suited for field conditions and convenient transportation of samples back to the laboratory. Thus, saliva can be considered a reliable source of DNA for a wide variety of genetic studies. [Copyright &y& Elsevier]