Evaluation of fecal DNA preservation techniques and effects of sample age and diet on genotyping success

Optimal collection and preservation protocols for fecal DNA genotyping are not firmly established. We evaluated 3 factors that influence microsatellite genotyping success of fecal DNA extracted from coyote (Canis latrans) scats: 1) age of scat, 2) preservative, and 3) diet content. We quantified genotyping success by comparing rates of allelic dropout, false alleles, and failed amplifications among consensus genotypes. We used a panel of 6 microsatellite loci to genotype 20 scat samples, each of which was subjected to 3 age (1 day, 5 days, and 10 days post-deposition) and 3 preservation (DET buffer, 95% ethanol [EtOH], and lysis buffer) treatments. Both sample age and storage buffer had a significant effect on success and reliability. Ethanol and DET buffer preserved fecal samples with similar efficiency, and both were superior to lysis buffer. Our analysis of DNA degradation rates revealed that samples collected as early as 5 days of age yielded DNA that was highly degraded relative to samples collected on day 1. We tested the influence of dietary remains on microsatellite genotyping by using scat samples consisting predominantly of insect prey (n = 5), mammalian prey (n = 9), or the remains of juniper (Juniperus spp.) berries (n = 6) and compared EtOH and DET buffer preservation efficacy. We observed a significant interaction effect between storage buffer and diet for the probability of a false allele in a polymerase chain reaction (PCR), suggesting that the optimal preservation technique depended on the food remains comprising the scat. Scats comprised of juniper berry remains were more reliably genotyped when preserved in DET than EtOH. Mammalian prey-based scats were more reliable when stored in EtOH than DET buffer. Insect-predominant scats were preserved in EtOH and DET buffer with similar efficiency. Although accurate and reliable results can be obtained from scats collected at ≥5 days of age, we suggest sampling design to include collection of scats <5 days of age to minimize field and laboratory expenses. We suggest EtOH preservation for scats of obligate carnivores and of facultative carnivores with a diet consisting primarily of mammals. We suggest DET buffer preservation for animals with a diet consisting of plant-derived foods. Lysis buffer protocols that we employed should not be used for fecal DNA preservation. © 2011 The Wildlife Society.     

Propylene glycol: a promising preservative for insects,comparable to ethanol,from trapping to DNA analysis

Recent advances in DNA analysis allow us to identify an unprecedented number of insect samples collected by mass sampling techniques such as insect traps. In these circumstances, a preservative that can be applied from trap to storage is necessary to prevent degradation of DNA before analysis and save on the cost of labor for collecting insects from traps. Propylene glycol has a prominent feature as a trap solution. We aimed to examine the DNA preservability of 98% propylene glycol at 2 weeks and more than 6 months after initial collection in comparison with 99.5% ethanol, which is commonly used for storage of specimens for genetic analysis. We compared amplification performance of PCR targeting a specific region of the mitochondrial cytochrome c oxidase subunit I (COI) gene in the orders Hymenoptera, Diptera, and Coleoptera using two extraction methods varying in extraction efficiency. Even after 6 months, more than 75% of samples were recognized to have succeeded in PCR amplification irrespective of preservatives by the extraction method with higher extraction efficiency. It suggested that mitochondrial DNA was preserved in both solutions. However, dim bands in the electrophoreses of PCR products increased with time in extracts by another method with lower extraction efficiency. In Diptera and Coleoptera, the rate of dim bands increased more rapidly for ethanol-preserved than for propylene glycol-preserved specimens, indicating higher DNA preservability of propylene glycol over time for these taxa. On the other hand, in Hymenoptera, the preservatives did not affect PCR amplification performance. Considering its safer characteristics and high DNA preservability in a wide range of taxa, propylene glycol can be a promising solution from trapping of insects to storage for genetic analysis.     

Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus Dalbergia

Five published DNA extraction protocols were compared for their ability to produce good quality DNA from fresh and herbarium leaves of several species of the genus Dalbergia. The leaves of these species contain high amounts of secondary metabolites, which make it difficult to perform a clean DNA extraction and thereby interfering with subsequent PCR amplification. The protocol that produced the best DNA quality in most of the Dalbergia species analyzed, utilizes polyvinylpyrrolidone to bind the phenolic compounds, a high molar concentration of NaCl to inhibit co-precipitation of polysaccharides and DNA, and LiCl for removing RNA by selective precipitation. The DNA quality of herbarium specimens was worse than that for fresh leaves, due to collecting conditions and preservation of samples. We analyzed 54 herbarium specimens, but the recovered DNA allowed successful PCR amplification in only eight. For the genus Dalbergia, the herbarium is an important source of material for phylogenetic and evolutionary studies; due to the occurrence of the different species in various geographical regions in Brazil, it is difficult to obtain fresh material in nature. Our results demonstrated that for Dalbergia species the methods used for the collection and preservation of herbarium specimens have a mayor influence on DNA quality and in the success of phylogenetic studies of the species.     

Isolation of DNA from museum exhibits of butterflies (Lepidoptera, Papilionidae) and PCR analysis with random and universal genes-specific primers

The effect of the duration of storage of entomological material on DNA preservation was estimated. The results of the optimization of conditions for the analysis of random amplified polymorphic DNA in a polymerase chain reaction (RAPD-PCR) are presented as applied to the DNA of lepidopterans of the family Papilionidae. RAPD patterns are shown for the first time in Atrophaneura alcinous and four species of the genus Parnassius (sensu lata). The applicability of museum specimens of butterflies for RAPD analysis was demonstrated. The results of PCR analysis using DNA obtained from different collection specimens stored for up to five years were compared. The authenticity of DNA obtained from collection specimens was proved using PCR with universal primers, which are specific to the COI and COII cytochrome genes of mitochondrial DNA (mt DNA). The lengths of individuals gene fragments obtained by the amplification of both museum and live specimens were 800 and 1600 bp. The conservative regions of mitochondrial genome were shown to be slightly different in two A. alcinous subspecies.     

How useful is DNA extracted from the legs of archived insects for microsatellite-based population genetic analyses?

DNA obtained from museum specimens provides a historical perspective on levels of genetic diversity. Archived samples are irreplaceable so it is desirable that only parts of the specimens are used, which constrains the amount of DNA obtained from small taxa. However, at present there are no quantitative data on yields of DNA from such samples. In this paper we determine the amount of DNA that may be extracted from the legs of museum-archived specimens of the damselfly Coenagrion mercuriale (Charpentier) and the suitability of this DNA for PCR-amplification of nuclear genetic loci (microsatellites). We find that (i) the yield of DNA correlates with the genotyping success rate and (ii) the amount of DNA obtained from the legs decreases with time since sample collection until 1954, before which no DNA could be detected (although DNA may be present in very low quantities). This cut-off point for successful DNA extraction corresponds with the date until reliable genotypes could be obtained by routine PCR. Thus, air-dried insect legs more than 50 years old appear to have limited usefulness for studies that seek to amplify many nuclear loci without the use of other techniques that may be used to increase the possible low-quantities of template DNA present.     

Salt drying: a low‐cost,simple and efficient method for storing plants in the field and preserving biological repositories for DNA diversity research

Although a variety of methods have been optimized for the collection and storage of plant specimens, most of these are not suited for field expeditions for a variety of logistic reasons. Drying specimens with silica gel in polyethylene bags is currently the standard for field‐sampling methods that are suitable for subsequent DNA extraction. However, silica‐gel repositories are not readily available in remote areas, and its use is not very cost‐effective for the long‐term storage of collections or in developing countries with limited research budgets. Salting is an ancient and traditional drying process that preserves food samples by dehydrating tissues and inhibiting water‐dependent cellular metabolism. We compared salt and silica‐gel drying methods with respect to dehydration rates overtime, DNA quality and polymerase chain reaction(PCR) success to assess whether dry salting can be used as an effective plant preservation method for DNA analysis. Specimens from eleven plant species covering a variety of leaf structures, leaf thicknesses and water contents were analysed. Experimental work indicated that (i) levels of dehydration in sodium chloride were usually comparable to those obtained when silica gel was used, (ii) no spoilage, fungal or bacterial growth was observed for any of the species with all drying treatments and (iii) good yields of quality genomic DNA suitable for PCR applications were obtained in the salt‐drying treatments. The preservation of plant tissues in commercial table salt appears to be a satisfactory, and versatile method that may be suitable in remote areas where cryogenic resources and silica repositories are not available.     

Field preservation of monogenean parasites for molecular and morphological analyses

In the present work we examined the efficacy of three different chemical solutions (EtOH 70%, DMSO-NaCl solution, and Longmire buffer) in field preservation of fish gills to be subsequently screened for monogenean specimens destined to morphological and molecular analyses. Degree of difficulty in collecting monogeneans from gills, morphological state of parasites, integrity of their DNA and reliability of sequence reading were observed and qualitatively compared to those of gills and parasites stored in 5% formalin and 99% ethanol. Data were collected over a period of 2 months. Storage in Longmire buffer resulted in dissociation of gills and parasites, while both DMSO and 70% ethanol provided a fine physical and molecular preservation of gills and monogeneans, allowing rapid collection of parasites from lamellae, and easy extraction, amplification and sequencing of parasitic DNA.     

Evaluation of mailed pediatric buccal cytobrushes for use in a case-control study of birth defects

BACKGROUND: Buccal cell collection is a convenient DNA collection method; however, little attention has been given to the quality of DNA obtained from pediatric populations. The purpose of this study was to determine the effect of a modified cytobrush collection method on the yield and quality of infant buccal DNA collected as part of a population-based case-control study of birth defects. METHODS Cytobrushes were collected from infants, mothers, and fathers using a standard collection method in 1997 to 2003 and a modified protocol that allows air-drying of the cytobrushes after collection from 2003 to the present. Yield and quality of DNA from 1057 cytobrushes was assessed by quantitative PCR and short tandem repeat (STR) genotyping, respectively. RESULTS Air-dried cytobrushes from infants had higher median DNA yields (1300 ng) and STR completion rates (99.5%) than standard collection method cytobrushes (60 ng and 59.5%, respectively). A subset of DNA aliquots was genotyped for six single nucleotide polymorphisms (SNPs). Aliquots from both collection methods that passed the quality protocol (DNA concentration >1 ng/μl, and successful amplification of ≥1 STR) had high genotype completion rates (99-100%). The median DNA yield following whole genome amplification was more than twofold higher for air-dried than standard collection specimens (p < 0.001). CONCLUSION Yield and quality of buccal DNA collected from infants are improved by using a method that incorporates air-drying; however, DNA collected by both methods is suitable for genotyping if stringent quality control procedures are instituted. These findings may be helpful for future epidemiologic studies of birth defects and other adverse pediatric outcomes.     

How old can we go? Evaluating the age limit for effective DNA recovery from historical insect specimens

Historical museum specimens are valuable for exploring population genetics and evolutionary questions because they can provide snapshots of morphological and genetic characteristics from populations over space and time. Unfortunately, DNA found in older museum specimens is frequently degraded, so obtaining genotypes from many individual samples necessary for rigorous molecular population genetic studies is challenging. Previous studies have varied greatly in their success at obtaining genotypes from older preserved insect material. Many well-intentioned collection curators have used research results showing poor preservation of DNA preserved in museum specimens to inform curatorial best practices, in some cases choosing not to allow DNA extraction by destructive sampling because, in their estimation, the likelihood of success would be low. Recent methodological advances in DNA extraction, amplification, and genotyping have allowed some researchers to include mid-19th century samples in molecular genetic analyses. Here we present a robust, high-throughput, and low-cost DNA extraction and genotyping protocol for historical insect specimens employing restriction digests of PCR products followed by high sensitivity electrophoresis. Using this technique, we obtained mitochondrial haplotypes for 100% of 48 New World Junonia butterfly specimens (Nymphalidae) ranging in age from pre-1813 to 1909 and show that the haplotype frequencies obtained are statistically indistinguishable from 20th-century and contemporary reference populations of Junonia (1632 specimens) matched by geographic region. As most extant insect specimens were collected after 1813, based on our findings we would expect that many or even most pinned specimens preserved in museum collections contain usable DNA for mitochondrial haplotyping.     

Ancient DNA preserved in small bone fragments from the P.W. Lund collection

The Lund collection is one of the oldest subfossil collections in the world. The vast assemblage of subfossils was collected in the 1830s and 1840s by Peter Wilhelm Lund in Lagoa Santa, Brazil, and was shipped to Copenhagen in 1848, where it was stored in various locations around the city with little attention for the future preservation of the collection. So far, successful genetic research on the material collected by Lund has been limited to two samples of human petrous bone. However, less is known about the preservation conditions of the vast amounts of small and fragmentary bones stored in the collection. To address this, we studied ancient DNA from bulk bone samples of approximately 100 bone fragments from the P.W. Lund collection from boxes with varying degrees of physical preservation conditions. Using bulk bone metabarcoding, we found a high species diversity in all samples. In total, we identified 17 species, representing 11 mammals, two birds, one fish, and three frogs. Of these, two species are new to the collection. Collectively, these results exhibit the potential of future genetic studies on the famous P.W. Lund collection and suggest that the effects of poor storage conditions are probably negligible compared with the long‐term in situ degradation that specimens undergo before excavation.     

Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.     

Genotyping of urinary samples stored with EDTA for forensic applications

Individual identification of urinary samples is necessary when sample switching or handling are suspected during a judicial process. To improve the rate of successful genotyping of urinary samples, we examined the stability of DNA in urinary samples stored for up to 30 days. Urinary samples from 20 healthy individuals (10 males and 10 females) were stored at -80°C with different concentrations of EDTA (0, 10 and 40 mM). Urinary DNA was extracted at days 0, 3, 9, and 30 after collection. The Quantifiler Human DNA Quantification Kit was used for measuring DNA concentration. Twenty STR loci were co-amplified using amelogenin-specific PCR with the Goldeneye 20A Kit. Significant differences in DNA concentration were observed between samples from females and males. In the case of female urinary DNA preservation with 10 and 40 mM EDTA the mean detection rate reached 0.95 after up to 30 days; for the male urinary samples, the mean detection rate of urinary DNA preserved with 40 mM EDTA was significantly higher than with 10 mM. We concluded that 40 mM EDTA is the best concentration for preservation of the DNA in urinary samples.     

Comparison of destructive and nondestructive DNA extraction methods for the metabarcoding of arthropod bulk samples

DNA metabarcoding is routinely used for biodiversity assessment, in particular targeting highly diverse groups for which limited taxonomic expertise is available. Various protocols are currently in use, although standardization is key to its application in large-scale monitoring. DNA metabarcoding of arthropod bulk samples can be conducted either destructively from sample tissue, or nondestructively from sample fixative or lysis buffer. Nondestructive methods are highly desirable for the preservation of sample integrity but have yet to be experimentally evaluated in detail. Here, we compare diversity estimates from 14 size-sorted Malaise trap samples processed consecutively with three nondestructive approaches (one using fixative ethanol and two using lysis buffers) and one destructive approach (using homogenized tissue). Extraction from commercial lysis buffer yielded comparable species richness and high overlap in species composition to the ground tissue extracts. A significantly divergent community was detected from preservative ethanol-based DNA extraction. No consistent trend in species richness was found with increasing incubation time in lysis buffer. These results indicate that nondestructive DNA extraction from incubation in lysis buffer could provide a comparable alternative to destructive approaches with the added advantage of preserving the specimens for postmetabarcoding taxonomic work but at a higher cost per sample.     

A factorial design experiment as a pilot study for noninvasive genetic sampling

Noninvasive genetic sampling has increasingly been used in ecological and conservation studies during the last decade. A major part of the noninvasive genetic literature is dedicated to the search for optimal protocols, by comparing different methods of collection, preservation and extraction of DNA from noninvasive materials. However, the lack of quantitative comparisons among these studies and the possibility that different methods are optimal for different systems make it difficult to decide which protocol to use. Moreover, most studies that have compared different methods focused on a single factor – collection, preservation or extraction – while there could be interactions between these factors. We designed a factorial experiment, as a pilot study, aimed at exploring the effect of several collection, preservation and extraction methods, and the interactions between them, on the quality and amplification success of DNA obtained from Asiatic wild ass (Equus hemionus) faeces in Israel. The amplification success rates of one mitochondrial DNA and four microsatellite markers differed substantially as a function of collection, preservation and extraction methods and their interactions. The most efficient combination for our system integrated the use of swabs as a collection method with preservation at ?20 °C and with the Qiagen DNA Stool Kit with modifications as the DNA extraction method. The significant interaction found between the collection, preservation methods and the extraction methods reinforces the importance of conducting a factorial design experiment, rather than examining each factor separately, as a pilot study before initiating a full‐scale noninvasive research project.     

Utilizing field collected insects for next generation sequencing: Effects of sampling,storage, and DNA extraction methods

DNA sequencing technologies continue to advance the biological sciences, expanding opportunities for genomic studies of non‐model organisms for basic and applied questions. Despite these opportunities, many next generation sequencing protocols have been developed assuming a substantial quantity of high molecular weight DNA (>100 ng), which can be difficult to obtain for many study systems. In particular, the ability to sequence field‐collected specimens that exhibit varying levels of DNA degradation remains largely unexplored. In this study we investigate the influence of five traditional insect capture and curation methods on Double‐Digest Restriction Enzyme Associated DNA (ddRAD) sequencing success for three wild bee species. We sequenced a total of 105 specimens (between 7–13 specimens per species and treatment). We additionally investigated how different DNA quality metrics (including pre‐sequence concentration and contamination) predicted downstream sequencing success, and also compared two DNA extraction methods. We report successful library preparation for all specimens, with all treatments and extraction methods producing enough highly reliable loci for population genetic analyses. Although results varied between species, we found that specimens collected by net sampling directly into 100% EtOH, or by passive trapping followed by 100% EtOH storage before pinning tended to produce higher quality ddRAD assemblies, likely as a result of rapid specimen desiccation. Surprisingly, we found that specimens preserved in propylene glycol during field sampling exhibited lower‐quality assemblies. We provide recommendations for each treatment, extraction method, and DNA quality assessment, and further encourage researchers to consider utilizing a wider variety of specimens for genomic analyses.     

Lessons from genome skimming of arthropod‐preserving ethanol

Field‐collected specimens of invertebrates are regularly killed and preserved in ethanol, prior to DNA extraction from the specimens, while the ethanol fraction is usually discarded. However, DNA may be released from the specimens into the ethanol, which can potentially be exploited to study species diversity in the sample without the need for DNA extraction from tissue. We used shallow shotgun sequencing of the total DNA to characterize the preservative ethanol from two pools of insects (from a freshwater habitat and terrestrial habitat) to evaluate the efficiency of DNA transfer from the specimens to the ethanol. In parallel, the specimens themselves were subjected to bulk DNA extraction and shotgun sequencing, followed by assembly of mitochondrial genomes for 39 of 40 species in the two pools. Shotgun sequencing from the ethanol fraction and read‐matching to the mitogenomes detected ~40% of the arthropod species in the ethanol, confirming the transfer of DNA whose quantity was correlated to the biomass of specimens. The comparison of diversity profiles of microbiota in specimen and ethanol samples showed that ‘closed association’ (internal tissue) bacterial species tend to be more abundant in DNA extracted from the specimens, while ‘open association’ symbionts were enriched in the preservative fluid. The vomiting reflex of many insects also ensures that gut content is released into the ethanol, which provides easy access to DNA from prey items. Shotgun sequencing of DNA from preservative ethanol provides novel opportunities for characterizing the functional or ecological components of an ecosystem and their trophic interactions.     

DNA microarray probe preparation by gel isolation nested PCR

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.     

聚合酶链式反应检测结核杆菌的研究

以人型结核杆菌基因组DNA为模板,合成二段引物各20个碱基进行聚合酶链式反应(PCR)。经琼脂糖凝胶电泳证实,获得一条245bp扩增带。PCR检测的敏感性染色体基因组DNA为1pg,菌悬液为13个活菌／ml。在特异性试验中,人型结核杆趋,牛型结核杆菌、BCG可见此扩增带。被试的其它14种扰酸菌以及变铅青链霉菌、大肠杆菌质粒Puc19、星状诺卡氏菌、红球菌均未见该扩增带。54例肺结核痰标本3种方法检查的阳性率分别为：萋尼氏抗酸染色16．7％,培养法14．8％,PCR 37．0％。前2种检查方法分别与PCR比较,经统计学处理均有显著性差异(P<0．01)。12例非结核性肺部疾患痰标本抗酸染色和PCR均为阴性。结果表明,PCR技术是快速、敏感、特异诊断结核病的方法。     

Large-scale general collection of wild-plant DNA in Mustang, Nepal

The deposit of DNA samples of wild plants that correspond to voucher specimens is highly informative and greatly enhances the value of the herbarium specimens. The Society of Himalayan Botany (SHB), Tokyo, has assembled general collections of flowering plants of the Sino-Himalayan region for more than 40 years. In a trial of the collection of these types of bioresources for use in basic research, we adopted FTA cards, which have recently been used for large-scale collection of DNA of humans, microorganisms and viruses, for the general collection of DNA samples of wild plants during a botanical expedition in Mustang, Nepal, in 2003. Three hundred and fifty-five plant specimens from Mustang, Nepal, were collected along with the corresponding DNA samples. Examination of the quality of the DNA samples by PCR demonstrated the utility of the collection system. The identification of all of the specimens collected, as well as data from the specimens, will be presented on the Flora of Nepal Database website (), which is open to the public. The DNA resources will be identified on the website and distributed openly by the SHB to researchers worldwide for basic research.     

Evaluation of buccal cell collection protocols for genetic susceptibility studies

Buccal cells are increasingly used as a source of quality DNA to improve participation rates in molecular studies. Here, three buccal cell collection protocols were compared to determine factors affecting the yield of cells, total DNA per sample, and DNA yield per cell. In addition, kinetic quantitative polymerase chain reaction (PCR) (TaqMan™) was used to quantify human DNA available for PCR. The method of collection used influenced the overall DNA yield per sample. The collection buffer used influenced the number of cells but not the overall DNA yield per sample. Repeated freezing and thawing did not affect overall DNA yield per sample, DNA yield per cell, or the total number of cells collected. Mouthwashes had the highest DNA yield per sample (20.8 µg) compared with cytobrush samples (1.9 µg from three cytobrushes) and tongue depressors (0.8 µg from three tongue depressors). However, mouthwash samples may contain significant non-human DNA and other contaminants that could interfere with some molecular studies. Spectrometry grossly overestimated the total DNA recovered from mouthwash samples compared with fluorometry or quantitative PCR.