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Background. High content of organic compounds that worsen nucleic acid purification in aromatic plants, as well as the use of such toxic substances as phenol and mercaptoethanol in many protocols for plant DNA isolation, make it advisable to take the above disadvantages into account when optimizing the DNA extraction technique for the work with essential oil rose plants. Material and methods. Rose accessions from the Crimean Federal University’s Botanical Garden, and those from the collection held by the Research Institute of Agriculture of Crimea were included in the study. DNA extraction was done according to a modified CTAB protocol. Effectiveness of the technique was assessed using spectrophotometry, horizontal electrophoresis, and ISSR-PCR. Results. DNA preparations extracted with the modified technique were well visualized on the electropherogram and demonstrated high spectrophotometric values. DNA content was twice as high in preparations isolated with an extraction buffer with PVP, compared to a PVP-free buffer. The concentration was also higher in DNA extracts from stems than that from leaves. Purity parameters expressed by the absorption ratios at wavelengths A260/280 and A260/230 were again higher for DNA extracts from stems isolated with an enriched buffer, the A260/230 ratio falling within the normal range only in DNA extracted from stems in the presence of PVP. Besides, DNA extracts were effectively purified from proteins without phenol or mercaptoethanol, due to double rinsing with a chloroform/isoamyl alcohol (24 : 1) mixture. Conclusion. Using rose stem tissues as the research material, adding 2% PVP to the extraction buffer, and twofold rinsing with a chloroform/isoamyl alcohol mixture made it possible to obtain DNA extracts with high concentrations and purity indices within normal ranges suitable for the ISSR analysis of essential oil rose genetic diversity.

Large-scale genotyping platforms are currently being developed for several wild species. By querying thousands of polymorphic loci, genomics can be a useful ecological tool for describing and monitoring populations. Genomics is becoming increasingly useful as a forensic tool because of its ability to identify population of origin for purposes of enforcing anti-poaching laws. Our aim was to test the new SNP chip for caribou/reindeer (Rangifer tarandus) (Illumina iSelect caribou 60 K) under recommended and non-optimal sample conditions. Impact on signal detection (call rate) and error rate were assessed using reference samples. The SNP chip was shown to be robust, highly sensitive, reliable, and accurate at more than 10-fold below the recommended DNA input. Biological source of DNA had minor impact, even with fecal pellets given sufficient amount of host DNA. Hybridization of non-Rangifer samples as well as samples bearing DNA from two Rangifer samples both showed a drop in call rate and shifted levels of heterozygosity. Based on a population-targeted subset of SNPs included in the chip design, reassignment of 981 samples to a functional group (here to a caribou ecotype) was highly accurate (99.59 %) and the relative probability of reassignment error was estimated using the logarithm of odds score. Overall, the SNP chip is suitable for analysis of caribou/reindeer genomes even with suboptimal sampling and hence useful for population management and forensics.

DNA quality is of paramount importance for molecular biology research. This study aimed to assess the DNA extracted from residual blood clots after serological testing, focusing on the impact of blood clot segments, extraction kits, temporary storage durations (TSDs), and thawing methods on DNA quality. We divided the residual blood clot column (BCC) from healthy donors into three segments and utilized two different extraction kits. The BCCs were subjected to four TSDs at 4°C (7 days, 10 days, 1 month, and 2 months) and three thawing methods (4°C, room temperature, and 37°C). We found that the TIANamp Blood Clot DNA Kit yielded consistently high‐quality DNA from each segment with stable A260/280 and A260/230 ratios. The DNA yield showed a strong positive correlation with leukocyte concentration, and a satisfactory median DNA yield of 28.79 μg/g BCC was obtained across all segments. DNA integrity, as measured by the DNA integrity number and DNA fragment peak size, decreased with increasing TSD at 4°C, with a notable decrease after 10 days of storage. Thawing at 37°C resulted in the lowest DNA fragment peak size. In conclusion, BCC could be an ideal DNA source with satisfactory yield and purity. A prolonged TSD at 4°C leads to an obvious decrease in DNA integrity, and thawing the frozen BCC at 37°C decreases DNA fragment sizes. To maintain DNA integrity, BCCs should be cryopreserved as soon as possible after short TSDs at 4°C and thawed at 4°C. Significance statement: This study aimed to improve the DNA quality of residual blood clot column (BCC) after serological testing, as this material could be a valuable DNA source. However, few studies have investigated whether centrifugation or commercial extraction kits would affect the DNA productivity of BCC. In addition, the effect of storage time at 4°C and the thawing method of frozen BCC on the DNA quality of BCC varied in previous studies. The results of this research showed that satisfactory DNA could be obtained from each segment of BCC using the TIANamp blood clot DNA kit. However, the DNA integrity number values and DNA fragment sizes decreased with increasing temporary storage durations at 4°C and thawing the frozen BCC at 37°C resulted in the lowest DNA fragment sizes. An optimized scheme for BCC preservation and utilization was established in this research, which provides additional options for DNA sources. [ABSTRACT FROM AUTHOR]

Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing in clinical cancer care comes almost universally from formalin-fixed, paraffin-embedded tissue (FFPE). As newer technologies emerge for DNA characterization that requires higher molecular weight DNA, it was necessary to compare the quality of DNA in terms of DNA length between FFPE and cryopreserved samples. We hypothesized that cryopreserved samples would yield higher quantity and superior quality DNA compared to FFPE samples. We analyzed DNA metrics by performing a head-to-head comparison between FFPE and cryopreserved samples from 38 human tumors representing various cancer types. DNA quantity and purity were measured by UV spectrophotometry, and DNA from cryopreserved tissue demonstrated a 4.2-fold increase in DNA yield per mg of tissue (p-value < 0.001). DNA quality was measured on a fragment microelectrophoresis analyzer, and again, DNA from cryopreserved tissue demonstrated a 223% increase in the DNA quality number and a 9-fold increase in DNA fragments > 40,000 bp (p-value < 0.0001). DNA from the cryopreserved tissues was superior to the DNA from FFPE samples in terms of DNA yield and quality.

Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing in clinical cancer care comes almost universally from formalin-fixed, paraffin-embedded tissue (FFPE). As newer technologies emerge for DNA characterization that requires higher molecular weight DNA, it was necessary to compare the quality of DNA in terms of DNA length between FFPE and cryopreserved samples. We hypothesized that cryopreserved samples would yield higher quantity and superior quality DNA compared to FFPE samples. We analyzed DNA metrics by performing a head-to-head comparison between FFPE and cryopreserved samples from 38 human tumors representing various cancer types. DNA quantity and purity were measured by UV spectrophotometry, and DNA from cryopreserved tissue demonstrated a 4.2-fold increase in DNA yield per mg of tissue (p-value < 0.001). DNA quality was measured on a fragment microelectrophoresis analyzer, and again, DNA from cryopreserved tissue demonstrated a 223% increase in the DNA quality number and a 9-fold increase in DNA fragments > 40,000 bp (p-value < 0.0001). DNA from the cryopreserved tissues was superior to the DNA from FFPE samples in terms of DNA yield and quality. [ABSTRACT FROM AUTHOR]

This study was carried out to evaluate the effect of temperature during storage of maize leaves and extracted DNA on its quality and quantity in order to be efficiently amplified using PCR. Leaves were collected from the fourweek-old plants and divided into three groups of 20 samples. The first group of leaves was processed immediately, while the other two were stored at -20°C or - 80°C for 30 days. The DNA extracted from the fresh leaves was divided into three portions with the first being amplified immediately and the other two were stored at -20°C or -80°C for 30 days. The DNA quality and quantity were examined using a biospectrometer, after which the samples were diluted for the PCR assay. The quality of all DNA samples was at an acceptable level with their average OD260/280 ratio in the range from 1.85 to 1.87. The concentration of the DNA extracted immediately from fresh leaf tissue was not statistically different from the stored samples. Both the quality and quantity of DNA in all samples were sufficient for successful PCR amplification with two opaque2-specific molecular markers. Phi057 amplified a ~170bp fragment in QPM and ~160bp in non-QPM, while umc1066 amplified a ~150bp fragment in QPM and ~160-170bp in non-QPM. Our results suggest that appropriate storage conditions do not affect the DNA quality and quantity. This could be useful in marker-assisted selection of target genes, when a large number of samples must be processed prior to pollination, allowing breeders to discard plants lacking the desired alleles and reduce the size of the breeding population.

Abstract Background Genetic and genomic studies are seeing an increase in sample sizes together with a wider range of species investigated in response to environmental change concerns. In turn, these changes may come with challenges including the time and difficulty to isolate nucleic acids (DNA or RNA), the sequencing cost and environmental impacts of the growing amount of plastic waste generated in the process. Pseudotsuga menziesii var. menziesii (Mirbel) Franco (PM), Tsuga heterophylla (Raf.) Sarg. (TH) and Thuja plicata Donn ex D.Don (TP) are conifer species found in diverse woodlands both as natives and naturalized exotics. Our study was carried out whilst investigating their genetics to understand their population structure and potential for adaptation. Results In the present study, we compared two different DNA isolation methods, i.e., spin-column DNeasy plant mini kit (QIAGEN), and temperature-driven enzymatic cocktail Plant DNA Extraction (MicroGEM). The quantity of recovered DNA and the quality of DNA were assessed along with the plastic footprint and time needed for three tree species. Both methods were optimised and proven to provide enough DNA for each studied species. The yield of DNA for each method depended on the species: QIAGEN showed higher yield in P. menziesii and T. heterophylla, while T. plicata recovered similar amount of DNA for both methods. The DNA quality was investigated using DNA barcoding techniques by confirming species identity and species discrimination. No difference was detected in the PCR amplification of the two barcoding loci, (rbcL and trnH-psbA), and the recovered sequences between DNA isolation methods. Measurement of the plastic use and the processing time per sample indicated that MicroGEM had a 52.64% lower plastic footprint and was 51.8% faster than QIAGEN. Conclusions QIAGEN gave higher yields in two of the species although both methods showed similar quality results across all species. However, MicroGEM was clearly advantageous to decrease the plastic footprint and improve the time efficiency. Overall, MicroGEM recovers sufficient and reliable DNA to perform common downstream analyses such as PCR and sequencing. Our findings illustrate the benefits of research and efforts towards developing more sustainable methods and techniques to reduce the environmental footprint of molecular analyses.

Faecal samples are an important source of genetic information for studies of wild animals. The quality and quantity of faecal DNA can, however, be affected by different factors. Our goal was to compare different dung sample storage and DNA extraction methods to optimise the quality and quantity of DNA extracted from forest elephant dung samples. Dung samples (n = 132) of forest elephants were collected in a forest area in Côte d'Ivoire. Each faecal sample was preserved in 50 mL cryotubes using five different methods: Ethanol 70%, formalin 10%, Sodium acetate, acetic acid, and formalin (SAF), dimethyl sulfoxide, and Sodium chloride (20% DMSO), and silica gel. We used the Cetyl Trimethyl Ammonium Bromide (CTAB) cationic detergent and the phenol chloroform DNA extraction procedures for comparison. DNA was successfully obtained from all 132 faecal samples using both DNA extraction methods. Regarding the total DNA extracted independently from the storage buffers, the phenol chloroform method yields a mean DNA concentration (2163.93 ± 918.62 ng/μL) sevenfold higher than that obtained with the optimised CTAB method (338.15 ± 528.54 ng/μL). Our study indicates optimised faecal sample storage and DNA extraction procedures that can help implement conservation strategies for forest elephants. [ABSTRACT FROM AUTHOR]

Background: Genetic and genomic studies are seeing an increase in sample sizes together with a wider range of species investigated in response to environmental change concerns. In turn, these changes may come with challenges including the time and difficulty to isolate nucleic acids (DNA or RNA), the sequencing cost and environmental impacts of the growing amount of plastic waste generated in the process. Pseudotsuga menziesii var. menziesii (Mirbel) Franco (PM), Tsuga heterophylla (Raf.) Sarg. (TH) and Thuja plicata Donn ex D.Don (TP) are conifer species found in diverse woodlands both as natives and naturalized exotics. Our study was carried out whilst investigating their genetics to understand their population structure and potential for adaptation. Results: In the present study, we compared two different DNA isolation methods, i.e., spin-column DNeasy plant mini kit (QIAGEN), and temperature-driven enzymatic cocktail Plant DNA Extraction (MicroGEM). The quantity of recovered DNA and the quality of DNA were assessed along with the plastic footprint and time needed for three tree species. Both methods were optimised and proven to provide enough DNA for each studied species. The yield of DNA for each method depended on the species: QIAGEN showed higher yield in P. menziesii and T. heterophylla, while T. plicata recovered similar amount of DNA for both methods. The DNA quality was investigated using DNA barcoding techniques by confirming species identity and species discrimination. No difference was detected in the PCR amplification of the two barcoding loci, (rbcL and trnH-psbA), and the recovered sequences between DNA isolation methods. Measurement of the plastic use and the processing time per sample indicated that MicroGEM had a 52.64% lower plastic footprint and was 51.8% faster than QIAGEN. Conclusions: QIAGEN gave higher yields in two of the species although both methods showed similar quality results across all species. However, MicroGEM was clearly advantageous to decrease the plastic footprint and improve the time efficiency. Overall, MicroGEM recovers sufficient and reliable DNA to perform common downstream analyses such as PCR and sequencing. Our findings illustrate the benefits of research and efforts towards developing more sustainable methods and techniques to reduce the environmental footprint of molecular analyses. [ABSTRACT FROM AUTHOR]

Clinical autopsies are performed to reveal the process of the disease that caused patient death and validate the diagnosis and treatment decisions. In pediatric clinical autopsy, the feedback provided to bereaved families has a considerable social impact; however, pediatric diseases are diverse, which makes it difficult to elucidate them. Therefore, it is necessary to employ molecular biology techniques in addition to conventional methods. Formalin-fixed, paraffin-embedded (FFPE) tissues are routinely prepared. However, clinical autopsy FFPE tissue processing is not standardized, and it is unclear whether DNA from such tissues can be used for comprehensive genomic analysis. In this study, we evaluated the DNA quality of FFPE tissues from 15 recent autopsy cases at a single-center children’s hospital using quantitative polymerase chain reaction [PCR (Q129/Q41)] and nanoelectrophoresis (DNA integrity number (DIN)). Good quality DNA was obtained from every organ type excluding bone marrow within 6 days of formalin fixation. Prolonged proteinase K digestion (48 h > 24 h > 1 h) and thicker tissue sections (10 µm > 1 µm) improved Q129/Q41; however, 24 h fixed FFPE tissues showed better DNA quality. We propose an optimal and feasible workflow for storing short-term fixed FFPE tissues as DNA-preserved FFPE tissues for future comprehensive genomic searches.

Canned tuna is one of the most commonly consumed food products globally. Due to its high profit-abilityandtheincreasingdemandforit,fraudulentcannedtunaproductshavebecomeaseriousproblem. The traceability of fish species in packaged material and, in the case of highly processed forms, in canned products, has become impossible; therefore, canned tuna is on the list of the top ten food items affected by fraud. These fraudulent actions cause not only unfair trade in the commer-cial market and fishing industry, but also cause health damage (such as allergies and poisoning) to the public. Complex food matrices also affect the extracted DNA quality when the main food products are served with another medium. Brine solutions, different kind of oil, and several types of sauce are used as filling medium in the canned tuna production process. These filling medium can cause con-tamination depending on whether they include oil, salt or other ingredients during DNA extraction from main products. DNA-based protocols have become popular due to their higher reliability rate compared to other protocols. This research investigates the potential impact of can-filling medium on DNA quality, which is a key factor for food traceability research. With this aim, canned tuna from variousbrandsindifferentcan-fillingmediumsuchasoliveoil,sunfloweroilanddifferentkindsofsauces, were obtained from a Turkish supermarket. The quality properties, such as yield and purity, affected the traceability analyses. This study was designed to investigate the potential effect of the fillingmediumonDNAquality.Theresultsrevealedthatdifferentkindsofsauceutilizationasacan-filling medium cause a reduction in the DNA quality of canned tuna compared to other canned tunasamplesthatcontainoliveoilandsunfloweroil.ThepurityofextractedDNAincannedtunawhereoliveoilwasusedwasfoundtoberelativelyhigherthanothertunagroupswithdifferentcan-filling medium. Melting curve analyses revealed that sunflower oil causes relatively lower degra-dationthanoliveoilanddifferenttypesofsauceusedasfillingmedium.Theseresultscouldbebeneficial for further seafood traceability research, especially in complex matrices.

The acquisition of high‐quality biospecimens and the appropriate handling of these materials are indispensable for successful clinical sequencing. We developed a cancer clinical sequencing system targeting 160 cancer genes: PleSSision‐Rapid. Through the PleSSision‐Rapid system, we have analyzed DNA quality evaluated by DIN (DNA integrity number) with 1329 formalin‐fixed paraffin embedded (FFPE) samples including 477 prospectively collected tissues for genomic test (P) and 852 archival samples after routine pathological diagnosis (A1/A2). As a result, the samples with more than DIN 2.1 was 92.0% (439/477) in prospectively collected sample (P), while it was 85.6% (332/388) and 76.7% (356/464) in two types of archival samples (A1/A2). We performed the PleSSision‐Rapid sequence using the samples with over DIN 2.1 and DNA concentration >10 ng/μL with which we were able to construct a DNA library, and the probability of sequence success was almost equivalent during all types of specimen processing, at 90.7% (398/439) in (P), 92.5% (307/332) in (A1) and 90.2% (321/356) in (A2), respectively. Our result indicated the clinical benefit to prepare the prospective collection of FFPE materials for indisputable clinical sequence, and that DIN ≥ 2.1 would be a solid parameter for sample preparation of comprehensive genomic profiling tests. [ABSTRACT FROM AUTHOR]

The human milk microbiota (HMM) of healthy women can vary substantially, as demonstrated by recent advances in DNA sequencing technology. However, the method used to extract genomic DNA (gDNA) from these samples may impact the observed variations and potentially bias the microbiological reconstruction. Therefore, it is important to use a DNA extraction method that is able to effectively isolate gDNA from a diverse range of microorganisms. In this study, we improved and compared a DNA extraction method for gDNA isolation from human milk (HM) samples to commercial and standard protocols. We evaluated the extracted gDNA using spectrophotometric measurements, gel electrophoresis, and PCR amplifications to assess its quantity, quality, and amplifiability. Additionally, we tested the improved method's ability to isolate amplifiable gDNA from fungi, Gram-positive and Gram-negative bacteria to validate its potential for reconstructing microbiological profiles. The improved DNA extraction method resulted in a higher quality and quantity of the extracted gDNA compared to the commercial and standard protocols and allowed for polymerase chain reaction (PCR) amplification of the V3–V4 regions of the 16S ribosomal gene in all the samples and the ITS-1 region of the fungal 18S ribosomal gene in 95% of the samples. These results suggest that the improved DNA extraction method demonstrates better performance for gDNA extraction from complex samples such as HM. [ABSTRACT FROM AUTHOR]

Genetic methods for species identification are becoming increasingly popular and can accelerate insect monitoring. However, obtaining good DNA quality and quantity from insect traps remains a challenge for field studies. Ethylene glycol, propylene glycol, and Renner solution have been previously suggested as suitable preservatives for the collection of genetic material, but a systematic overview of their performance under compromising field conditions is lacking. Here we experimentally test whether and how different preservatives affect DNA quality under different conditions and evaluate how choice of preservative may affect metabarcoding and more demanding downstream applications (e.g., RADseq). For this, we used the house cricket, Acheta domesticus (L.) (Orthoptera: Gryllidae), and tested propylene glycol, ethylene glycol, and Renner solution for their ability to preserve DNA over 27 days in various dilutions and temperatures. DNA quality was measured as DNA fragmentation and success rates in PCR amplifying a COI fragment of 658, 313, or 157 bp. Undiluted propylene glycol and ethylene glycol always retained high molecular weight DNA at room temperature. No high molecular weight DNA was preserved at 37 °C or in any dilution. Nevertheless, the COI sequence could be amplified from samples at every condition. Renner solution did not preserve high molecular weight DNA and fragmentation increased over time at 37 °C until amplification was impossible. The results suggest that propylene glycol and ethylene glycol are suitable preservatives for collecting both genetic and morphological material, but dilution or high temperatures compromise their ability to preserve high molecular weight DNA. For genomic approaches requiring high DNA quality, additional preservatives may need to be tested. [ABSTRACT FROM AUTHOR]

ABSTRACT: As one of the main ingredients in some milk powders, whey powder is sometimes added to pure goat milk products, which can cause health risks, economic fraud, and unfair competition of food industries. This study is the first to explore qualitative and quantitative methods to identify adulteration of bovine whey powder in goat dairy products based on DNA. We extracted DNA from whey powder using a modified DNA extraction method; this exhibited good quality and integrity, with purity of 1.53 to 1.75 and concentration of 122 to 179 ng/μL. Conventional PCR and real-time PCR were compared for qualitative detection of bovine whey powder; real-time PCR demonstrated sensitivity of 0.01 ng/μL, which was higher than the 0.05 ng/μL detected by the conventional PCR method. Furthermore, real-time PCR was conducted for DNA quantitative detection, with good linearity (R2 = 0.9858) obtained for bovine whey powder contents from 0.1% to 30%. Relative error decreased with increase of the mixing proportion of whey powder; the coefficient of variation above 0.1% of the mixing ratio was close to or less than 5%; and the relative standard deviation of repeatability results was less than 5%. Considering the economic costs of testing, conventional PCR could be performed first, and samples with obvious intentional adulteration detected can be further accurately quantified by real-time PCR. Overall, this research provides a realistic and effective method for qualitative and quantitative identification of bovine whey powder in goat dairy products, thus laying a good foundation for verification of goat dairy product label claims and industrial control.

Microbial DNA extraction is a critical step in metagenomic research. High contents of chemical substances in wood tissues always cause low microbial DNA yield and quality. Up to date, almost no specialized methods involved in microbial DNA extraction from living wood were reported. In this study, an improved protocol (M1) concerning microbial DNA extraction from living poplar wood was developed. We compared microbial DNA yield and quality by M1 with those by other seven methods, including PowerSoil DNA isolation kit (M2), two soil microbial DNA extraction methods (M3 and M4), poplar genomic DNA extraction method from wood (M5), and microbial DNA extraction method from herb stems (M6), isolating bacteria (M7) and isolating fungus (M8). Results showed that M1 yielded much better quality and concentration of microbial DNA than the other methods (M2-M8) from both poplar wetwood and sapwood tissues. Following M1 protocol, 1 g of wetwood sample could yield 272.27 ng/ul (vol=50 ul) pure microbial DNA with the absorption ratios of 1.87 (A260/A230) and 1.66 (A260/A280). For 1 g of sapwood sample, these values were 361.83 ng/ul, 1.85 and 2.24, respectively. These DNA could be stably visualized by agarose gel electrophoresis and amplified by primer sets of bacteria (16S V3-V4, 16S-V4, 16S V4-V5) and fungus (ITS1, ITS2). While, the other seven methods only obtained less or contaminated microbial DNA, which could not be amplified stably by aforementioned primer sets. Our protocol provided an approach for microbial community study in living poplar wood in a more accurate way by molecular biology techniques.

Canned tuna is one of the most commonly consumed food products globally. Due to its high profitability and the increasing demand for it, fraudulent canned tuna products have become a serious problem. The traceability of fish species in packaged material and, in the case of highly processed forms, in canned products, has become impossible; therefore, canned tuna is on the list of the top ten food items affected by fraud. These fraudulent actions cause not only unfair trade in the commercial market and fishing industry, but also cause health damage (such as allergies and poisoning) to the public. Complex food matrices also affect the extracted DNA quality when the main food products are served with another medium. Brine solutions, different kind of oil, and several types of sauce are used as filling medium in the canned tuna production process. These filling medium can cause contamination depending on whether they include oil, salt or other ingredients during DNA extraction from main products. DNA-based protocols have become popular due to their higher reliability rate compared to other protocols. This research investigates the potential impact of can-filling medium on DNA quality, which is a key factor for food traceability research. With this aim, canned tuna from various brands in different can-filling medium such as olive oil, sunflower oil and different kinds of sauces, were obtained from a Turkish supermarket. The quality properties, such as yield and purity, affected the traceability analyses. This study was designed to investigate the potential effect of the filling medium on DNA quality. The results revealed that different kinds of sauce utilization as a can-filling medium cause a reduction in the DNA quality of canned tuna compared to other canned tuna samples that contain olive oil and sunflower oil. The purity of extracted DNA in canned tuna where olive oil was used was found to be relatively higher than other tuna groups with different can-filling medium. Melting curve analyses revealed that sunflower oil causes relatively lower degradation than olive oil and different types of sauce used as filling medium. These results could be beneficial for further seafood traceability research, especially in complex matrices. [ABSTRACT FROM AUTHOR]

Clinical autopsies are performed to reveal the process of the disease that caused patient death and validate the diagnosis and treatment decisions. In pediatric clinical autopsy, the feedback provided to bereaved families has a considerable social impact; however, pediatric diseases are diverse, which makes it difficult to elucidate them. Therefore, it is necessary to employ molecular biology techniques in addition to conventional methods. Formalin-fixed, paraffin-embedded (FFPE) tissues are routinely prepared. However, clinical autopsy FFPE tissue processing is not standardized, and it is unclear whether DNA from such tissues can be used for comprehensive genomic analysis. In this study, we evaluated the DNA quality of FFPE tissues from 15 recent autopsy cases at a single-center children's hospital using quantitative polymerase chain reaction [PCR (Q129/Q41)] and nanoelectrophoresis (DNA integrity number (DIN)). Good quality DNA was obtained from every organ type excluding bone marrow within 6 days of formalin fixation. Prolonged proteinase K digestion (48 h > 24 h > 1 h) and thicker tissue sections (10 µm > 1 µm) improved Q129/Q41; however, 24 h fixed FFPE tissues showed better DNA quality. We propose an optimal and feasible workflow for storing short-term fixed FFPE tissues as DNA-preserved FFPE tissues for future comprehensive genomic searches. [ABSTRACT FROM AUTHOR]

Study Question: What effects did treatment using hyaluronic acid (HA) binding/selection prior to ICSI have on clinical outcomes in the Hyaluronic Acid Binding sperm Selection (HABSelect) clinical trial?Summary Answer: Older women randomized to the trial's experimental arm (selection of sperm bound to immobilized (solid-state) HA) had the same live birth rates as younger women, most likely a result of better avoidance of sperm with damaged DNA.What Is Known Already: Recent randomized controlled trials (RCTs) investigating the efficacy of HA-based sperm selection prior to ICSI, including HABSelect, have consistently reported reductions in the numbers of miscarriages among couples randomized to the intervention, suggesting a pathological sperm-mediated factor mitigated by prior HA-binding/selection. The mechanism of that protection is unknown.Study Design, Size, Duration: The original HABSelect Phase 3 RCT ran from 2014 to 2017 and included 2752 couples from whom sperm samples used in control (ICSI) and intervention (Physiological IntraCytoplasmic Sperm Injection; PICSI) arms of the trial were stored frozen for later assessment of DNA quality (DNAq). The trial overlapped with its mechanistic arm, running from 2016 to 2018.Participants/materials, Setting, Methods: As miscarriage reduction was a significant secondary outcome of the trial, samples (n = 1247) selected for the mechanistic analysis were deliberately enriched for miscarriage outcomes (n = 92 or 7.4%) from a total of 154 miscarriages (5.6%) among all (n = 2752) couples randomized by stratified random sampling. Values from fresh semen samples for sperm concentration (mml), percentage forward progressive motility and percentage HA-binding score (HBS) were obtained before being processed by differential density gradient centrifugation or (rarely) by swim-up on the day of treatment. Surplus sperm pellets were recovered, aliquoted and cryopreserved for later analysis of DNAq using slide-based Comet, TUNEL, acridine orange (AO) and the sperm chromatin dispersion (SCD) assays. Following their classification into normal and abnormal sample subcategories based on reference values for sperm concentration and motility, relationships with HBS and DNAq were examined by Spearman correlation, Student's t-tests, Mann Whitney U tests, and logistic regression (univariable and multivariable). Parsimonious selection enabled the development of models for exploring and explaining data trends. Potential differences in future cumulative pregnancy rates relating to embryo quality were also explored.Main Results and the Role Of Chance: Results from the 1247 sperm samples assayed for HBS and/or DNAq, generated data that were considered in relation to standard physiological measures of (sperm) vitality and to treatment outcomes. All measures of HBS and DNAq discriminated normal from abnormal sperm samples (P < 0.001). SCD correlated negatively with the Comet (r = -0.165; P < 0.001) and TUNEL assays (r = -0.200; P < 0.001). HBS correlated negatively with AO (r = -0.211; P < 0.001), Comet (r = -0.127; P < 0.001) and TUNEL (r = -0.214; P < 0.001) and positively with SCD (r = 0.255; P < 0.001). A model for predicting live birth (and miscarriage) rates included treatment allocation (odds ratio: OR 2.167, 95% CI 1.084-4.464, P = 0.031), female age (OR 0.301, 95% CI 0.133-0.761, P = 0.013, per decade) and the AO assay (OR 0.79, 95% CI 0.60-1. 02.761, P = 0.073, per 10 points rise). A model predicting the expected rate of biochemical pregnancy included male age (OR 0.464, 95% CI 0.314-0.674, P < 0.001, per decade) and the SCD assay (OR 1.04, 95% CI 1.007-1.075, P = 0.018, per 10 point rise). A model for conversion from biochemical to clinical pregnancy did not retain any significant patient or assay variables. A model for post-injection fertilization rates included treatment allocation (OR 0.83, 95% CI 0.75-0.91, P < 0.001) and the Comet assay (OR 0.950, 95% CI 0.91-1.00, P = 0.041).Limitations, Reasons For Caution: HABSelect was a prospective RCT and the mechanistic study group was drawn from its recruitment cohort for retrospective analysis, without the full benefit of randomization. The clinical and mechanistic aspects of the study were mutually exclusive in that measures of DNAq were obtained from residual samples and not from HA-selected versus unselected sperm. Models for fitting mechanistic with baseline and other clinical data were developed to compensate for variable DNAq data quality. HABSelect used a solid-state version of PICSI and we did not assess the efficacy of any liquid-state alternatives. PICSI reduced fertilization rates and did not improve the outlook for cumulative pregnancy rates.Wider Implications Of the Findings: Notwithstanding the interventional effect on fertilization rates and possibly blastocyst formation (neither of which influenced pregnancy rates), poor sperm DNAq, reflected by lower HBS, probably contributed to the depression of all gestational outcomes including live births, in the HABSelect trial. The interventional avoidance of defective sperm is the best explanation for the equalization in live birth rates among older couples randomized to the trial's PICSI arm. As patients going forward for assisted conception cycles globally in future are likely to be dominated by an older demographic, HA-based selection of sperm for ICSI could be considered as part of their treatment plan.Study Funding/competing Interest(s): The study was supported by the National Institute for Health Research (NIHR) EME (Efficacy and Mechanism Evaluation)-11-14-34. National Research Ethics Service approval 11/06/2013: 13/YH/0162. S.L. is CEO of ExamenLab Ltd (company number NI605309).Trial Registration Number: ISRCTN99214271. [ABSTRACT FROM AUTHOR]

HLA sequence‐based DNA typing (SBT) by long‐range PCR amplification (LR PCR) and next‐generation sequencing (NGS) is a high‐throughput DNA sequencing method (LR‐NGS‐SBT) for the efficient and sensitive detection of novel and null HLA alleles to the field‐4 level of allelic resolution without phase ambiguity. However, the accuracy and reliability of the HLA typing results using buccal cells (BCs) and saliva as genetic source materials for the LR‐NGS‐SBT method are dependent largely on the quality of the extracted genomic DNA (gDNA) because a large degree of gDNA fragmentation can result in insufficient PCR amplification with the incorrect assignment of HLA alleles because of allele dropouts. In this study, we developed a new cost‐efficient swab storage gel (SSG) for wet swab collection of BCs (BC‐SSG) and evaluated its usefulness by performing different DNA analytical parameters including LR‐NGS‐SBT to compare the quality and quantity of gDNA extracted from BCs (in SSG or air dried), blood and saliva of 30 subjects. The BC‐SSG samples after 5 days of storage revealed qualitative and quantitative DNA values equivalent to that of blood and/or saliva and better than swabs that were only air‐dried (BC‐nSSG). Moreover, all the gDNA extracted from blood, saliva and BC‐SSG samples were HLA‐typed successfully to an equivalent total of 408 alleles for each sample type. Therefore, the application of BC‐SSG collection media for LR‐NGS‐SBT has benefits over BC dried samples (dry swabs) such as reducing retesting and the number of untestable BC samples because of insufficient DNA amplification. [ABSTRACT FROM AUTHOR]

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca), causal agents of citrus bacterial canker, are both regulated by the European Union to prevent their introduction. Xcc is responsible for severe outbreaks of citrus production worldwide, therefore, a prompt and reliable detection is advisable for the early detection of this bacterium either in symptomatic or asymptomatic plant material. The current EPPO (European and Mediterranean Plant Protection Organization) diagnostic protocol, PM 7/44(1), includes several diagnostic tests even if new assays have been developed in the latter years for which validation data are needed. Recently, a test performance study was organized within the Valitest EU Project to validate Xcc diagnostic methods and provide evidence on the most reliable assays; however, the influence of DNA extraction methods (DEM) on the reliability of the detection has never been assessed. In this study we evaluate four different DEM, by following two different approaches: (i) a comparison by real-time PCR standard curves of bacterial DNA versus bacterial DNA added to plant DNA (lemon, leaves and fruit; orange fruit); and (ii) the evaluation of performance criteria of spiked samples (plant extract added with ten-fold diluted bacterial suspensions at known concentrations). Droplet digital PCR is developed and compared with real-time PCR, as the detection method. [ABSTRACT FROM AUTHOR]

The human milk microbiota (HMM) of healthy women can vary substantially, as demonstrated by recent advances in DNA sequencing technology. However, the method used to extract genomic DNA (gDNA) from these samples may impact the observed variations and potentially bias the microbiological reconstruction. Therefore, it is important to use a DNA extraction method that is able to effectively isolate gDNA from a diverse range of microorganisms. In this study, we improved and compared a DNA extraction method for gDNA isolation from human milk (HM) samples to commercial and standard protocols. We evaluated the extracted gDNA using spectrophotometric measurements, gel electrophoresis, and PCR amplifications to assess its quantity, quality, and amplifiability. Additionally, we tested the improved method’s ability to isolate amplifiable gDNA from fungi, Gram-positive and Gram-negative bacteria to validate its potential for reconstructing microbiological profiles. The improved DNA extraction method resulted in a higher quality and quantity of the extracted gDNA compared to the commercial and standard protocols and allowed for polymerase chain reaction (PCR) amplification of the V3–V4 regions of the 16S ribosomal gene in all the samples and the ITS-1 region of the fungal 18S ribosomal gene in 95% of the samples. These results suggest that the improved DNA extraction method demonstrates better performance for gDNA extraction from complex samples such as HM.

DNA isolations from the whole blood are commonly performed to obtain DNA for molecular research and diagnostics. Generally, blood samples are taken into anticoagulant tubes and stored in deep freezers until DNA isolation. In fresh blood, pretreatments or leukocytes preparations can be performed and suggested for advanced DNA isolation. However, similar applications in freeze-thawed blood (FTB) have not been shown yet. In the study, centrifugation and washing of FTB were applied as pretreatment before DNA isolations, and their effects on isolated DNA characteristics including DNA integrity, quality, quantity, mitochondrial (mt), and nuclear (n) DNA levels were investigated. Microscopic and flow cytometric analyses were used to check leukocyte integrity in FTB. Spectrophotometric analysis was carried out to determine DNA quality and quantity in the isolated DNA samples. Real-time PCR analyses were used to check mtDNA/nDNA ratio and DNA integrity at the quantitative level. Cell integrity analyses showed that most of the leukocytes were intact in FTB. Therefore, centrifugation enabled intact leukocytes and nuclear pellets in FTB to be harvested and washed and could be applied as pretreatment before DNA isolations. PBS and water washing of FTB led to obtaining high-quality DNA without changing the nDNA/mtDNA ratio and DNA integrity. TE washing of FTB increased DNA quality and enriched nDNA level about 2-fold without changing DNA integrity. Centrifugation and harvesting of a higher volume of FTB increased isolated DNA yield and quality but decreased DNA integrity and nDNA level. To conclude, the pretreatments of FTB had the advantage to obtain DNA with high-quality and high-quantity and can be used before DNA isolation, but they may affect mtDNA/nDNA ratios and DNA integrity levels. The relevant pre-treatment used in the present study can be used and improved for desired DNA isolation from FTB samples. [ABSTRACT FROM AUTHOR]

The strawberry tree (Arbutus unedo) is well-known for the use of its leaves, fruits, bark and roots in traditional medicine and, more recently, in the therapy of hypertension, diabetes, inflammatory and cardiovascular diseases. The plant contains several antioxidant compounds. The diversity of natural A. unedo populations from Greece is studied with leaf morphometrics and DNA markers. Five natural populations spanning from east (Lesvos island 39°12′ N, 26°05′ E) to west (Igoumenitsa 39°30′ N, 20°15′ E) and from north (Arnea, 40°29′ N, 23°38′ E) to south (Ancient Olympia 37°38′ Ν, 21°46′ Ε) were sampled. The fifth population was that of Kassandreia (40°01′ N, 23°26′ E) and the average sample size per population was N = 20 trees. DNA extraction and isolation was a challenge due to high amounts of phenolics present in leaves (arbutin, catechin and ethyl gallate) and among the many protocols studied, the NucleoSpin® Plant II Mini Kit provided the best results for downstream applications. Morphometric population variation was studied by employing 11 leaf size and shape parameters recorded by image processing and analysing software. When contrasting north/south population comparisons, it was found that, regarding measurements of central tendency, the northern population (Kassandreia) presented the highest values, while in contrast, in the measures of spread, the highest values were found in the southern population (Ancient Olympia). Furthermore, statistically significant population differences were found in leaf size, but not in leaf shape parameters. The combination of DNA markers and morphometric analyses provides a foundation for diversity studies and characterization of A. unedo populations for downstream applications in population genetics studies, genetic conservation and in medicinal and natural products research.

The methods applied for DNA extraction from must and wine samples with monovarietal origin are presented and discussed aiming to prove the quality of extracted DNA and its good properties for further use in molecular tests. In the present research were compared four different DNA extraction methods from must and wine samples obtained from eleven V. vinifera varieties (five grapevine varieties for white wines and six grapevine varieties for red wines, respectively). Taking into consideration the amounts of obtained DNA, the concentrations and purities of the final DNA extracts, were stood out two modified methods. For all must samples, very efficient was the second method, which allowed obtaining a mean value of 87.9 ng µl-1 for the DNA concentration with 1.55 purity. Among the tested procedures, for monovarietal wine samples, the fourth method proved to be the most efficient which brought a mean value of 64.7 ng μl-1 for DNA concentration with 1.66 purity. This method adequate for wine samples involves two CTAB solution treatments and the RNase treatment applied before DNA resuspension. The DNA from must and wine extracts and the DNA from leaves of the corresponding grapevine varieties were amplified with five specific microsatellite primers (VVS2, VVMD27, VVMD32, VrZAG79 and VrZAG62). The aspects of pattern profiles were analysed in parallel and proved that the extracted DNA was suitable for amplification with these specific V. vinifera primers. The two selected extraction procedures are considered good for research purposes and ensure obtaining of good-quality extracted DNA from musts and one-year old wines.

Background: Microsatellite-based polymerase chain reaction (PCR) has great utility in microbial typing by analysis of band profiles of PCR amplicons. Bacterial DNA extraction methods affect yield and quality of DNA extract, and consequently subsequent molecular-based studies. The study aimed to evaluate impact of bacterial DNA extraction methods on band profiles of microsatellitebased PCR. Methods: Genomic DNA was extracted from Staphylococcus epidermidis ATCC 12228 using QIAamp kit, tween and SDS methods, and submitted to microsatellite-based PCR using (GACA)4 primer. For each extraction method, 4 concentrations were tested: the first crude extracted DNA and other 3 concentrations obtained by 1:1, 1:3, and 1:5 dilutions of the crude samples. Results: Yield and purity of DNA extracted by QIAamp kit were higher than those of other two methods. The PCR amplified all tested DNAs with generation of band profiles ranged from 2 to 11 bands in number, and from 200 bp to 1500 bp in size. Marked inter-method differences were found due to variation in band numbers and sizes. Band patterns obtained for QIAamp kit and tween methods were more robust than for SDS method. Variations of PCR patterns of different DNA concentrations within each method were minimal. Conclusions: Microsatellite-based PCR band profiles vary with different genomic DNA extraction methods for the same bacterial strain. Quality of extracted DNA is more influencing than its concentration. Therefore, it is recommended to unify the extraction method of bacterial DNA for all tested bacterial strains that are submitted to such type of PCR. [ABSTRACT FROM AUTHOR]

Abstract Background Breast cancer screening programs seem to be an insufficient tool for women at high genetic risk for breast cancer. These women are not adequately monitored yet. Genetic testing may improve clearly the quality of breast cancer prevention programs. At present, blood samples are favored for obtaining high-quality DNA; however, DNA can also be obtained by collecting saliva. The aim of this study was, on the one hand, to determine whether saliva sampling is a practicable means to obtain sufficient quantity and quality of DNA and, on the other hand, whether it is accepted by patients throughout mammographic diagnostics. Methods 67 consecutive women with diagnostic need for mammography with or without a family history for breast cancer were asked for their basic willingness to undergo a genetic testing by saliva sample in addition to standard diagnostics. Saliva samples were analyzed in terms of DNA quantity and quality. Results 64 (95.6%) women agreed to provide a saliva sample; 3 of them denied participation. And even 63 out of 64 (98.4%) were interested in their specific results. 45 out of 64 samples contained a DNA concentration above 50 ng/µl, 12 samples were between 25 and 50 ng/µl and only 7 of them were under 25 ng/µl with the standard extraction procedure. Conclusion A high number of patients seem to accept salvia samples as a risk assessment tool in breast diagnostics and are interested in their specific risk situation. At the same time, it could be demonstrated that it is an effective way to provide high-quality DNA for breast cancer gene analysis. However, it remains to be shown whether it would be possible to integrate it with the same acceptance in a nationwide breast cancer screening program.

The quality and quantity of DNA extracts used for genetic analysis are of utmost importance. The isolation of DNA from different types of samples can be done by classical methods or using specific kits, whose working methodology must strictly follow the protocol established by the manufacturer. The aim of this research was to evaluate the effectiveness of DNA extraction from 50 blood samples of Romanian Grey cows found in various genetic conservation programs due to their small number in cattle herds. The obtained results are considered satisfactory in terms of the purity of the obtained DNA extracts, with only one sample (2% of the total) having the DNA isolate contaminated with proteins based on the absorbance ratio A260/A280 lower than 1.8. [ABSTRACT FROM AUTHOR]

There is a growing interest in the tracking of genetic and epigenetic alterations in exhaled breath condensate (EBC) samples. The effects of different procedures on the quality and quantity of DNA in EBC were studied. The results demonstrated that sodium acetate precipitation and oligo (dT) improved the quality of the extracted DNA significantly (p < 0.01). Also, sodium acetate precipitation, using oligo (dT), incubation at 70 °C and SDS treatment increased the quantity of DNA significantly (p < 0.01). These results showed the advantages of the chemical and physical manipulations for improving the quality and quantity of the extracted DNA from EBC samples. [ABSTRACT FROM AUTHOR]

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The objects contained in crime scene and its surroundings have significance in the examination of forensic identification. The most commonly used specimens in the examination for identification are blood/blood spots, semen patches, vaginal swabs, buccal swabs and bones, including items often used by the perpetrator/victim the last time. For example, a mobile phone hearing aid (headset/earphone). In the use of earphones attached to the outer ear skin so it is suspected there is a serumen attached to the tool. One factor that can affect the quality of DNA is the prolonged exposure. Until now in Indonesia the effect of long exposure to room temperature on the quality of DNA on the DNA material of earphone swabs through DNA analysis has not been widely known. The type of study was laboratory experimental. Used earphones are exposed at room temperature within 1, 7, 14 and 20 days. The results of this study indicate that the environmental effect, ie the duration of exposure, affects the decrease in DNA content significantly from day 1 to 20. Detection of PCR mtDNA D-Loop HVS I visualization results 143 bp nt: 16268 16410 shows positive detection results (+) Only at day 1 exposure to room temperature [4 sample/66,67%] and day 7 [3 sample/50%]. Visualization of PCR results mtDNA D-Loop HVS II 126bp nt: 34 -159 was performed on day 1 day room exposure [2 sample/33,37%] and day 7 [6 sample/100%]. Conclusion, the duration of exposure to room temperature affect the quality of DNA from earpiece swab material. Decreased levels of earphones DNA swabs showed a significance value (p

Abstract Background Genomic DNA extracted from species of Cactaceae is often contaminated with significant amounts of mucilage and pectin. Pectin is one of the main components of cellular walls, whereas mucilage is a complex polysaccharide with a ramified structure. Thus, pectin- and mucilage-free extraction of DNA is a key step for further downstream PCR-based analyses. Results We tested our DNA extraction method on cladode tissue (juvenile, adult, and herbaria exemplars) of 17 species of Opuntia Mill., which are characterized by a large quantity of pectin and mucilage. Conclusion We developed a method for the extraction of gDNA free of inhibitory compounds common in species of Opuntia Mill., such as pectin and mucilage. Compared to previously extraction protocols, our method produced higher yields of high-quality genomic DNA.

Objectives: The aim of this study was to explore the variability in DNA quality and quantity along a gradient of industrial processing of botanical ingredients from raw materials to extracts. Methods: A data matrix was assembled for 1242 botanical ingredient samples along a gradient of industrial processing commonly used in the Natural Health Product (NHP) industry. Multivariate statistics was used to explore dependant variables for quality and quantity. The success of attaining a positive DNA test result along a gradient of industrial processing was compared among four biotechnologies: DNA barcoding, NGS, Sanger sequencing and qPCR. Results: There was considerable variance in DNA quality and quantity among the samples, which could be interpreted along a gradient from raw materials with greater quantities (50–120 ng/μL) of DNA and longer DNA (400-500bp) sequences to extracts, which were characterized by lower quantities (0.1–10.0 ng/μL) and short fragments (50-150bp). Conclusions: Targeted molecular diagnostic tests for species identity can be used in the NHP industry for raw and processed samples. Non-targeted tests or the use of NGS for any identity test needs considerable research and development and must be validated before it can be used in commercial operations as these methods are subject to considerable risk of false negative and positive results. Proper use of these tools can be used to ensure ingredient authenticity, and to avert adulteration, and contamination with plants that are a health concern. Lastly these tools can be used to prevent the exploitation of rare herbal species and the harvesting of native biodiversity for commercial purposes.

Background: Tumor mutational burden (TMB) measured via next‐generation sequencing (NGS)‐based gene panel is a promising biomarker for response to immune checkpoint inhibitors (ICIs) in solid tumors. However, little is known about the preanalytical factors that can affect the TMB score. Materials and Methods: Data of 199 patients with solid tumors who underwent multiplex NGS gene panel (OncoPrime), which was commercially provided by a Clinical Laboratory Improvement Amendments‐licensed laboratory and covered 0.78 megabase (Mb) of capture size relevant to the TMB calculation, were reviewed. Associations between the TMB score and preanalytical factors, including sample DNA quality, sample type, sampling site, and storage period, were analyzed. Clinical outcomes of patients with a high TMB score (≥10 mutations per megabase) who received anti‐programmed cell death protein 1 antibodies (n = 22) were also analyzed. Results: Low DNA library concentration (<5 nM), formalin‐fixed paraffin‐embedded tissue (FFPE), and the prolonged sample storage period (range, 0.9–58.1 months) correlated with a higher TMB score. After excluding low DNA library samples from the analysis, FFPE samples, but not the sample storage period, exhibited a marked correlation with a high TMB score. Of 22 patients with a high TMB score, we observed the partial response in 2 patients (9.1%). Conclusion: Our results indicate that the TMB score estimated via NGS‐based gene panel could be affected by the DNA library concentration and sample type. These factors could potentially increase the false‐positive and/or artifactual variant calls. As each gene panel has its own pipeline for variant calling, it is unknown whether these factors have a significant effect in other platforms. Implications for Practice: A high tumor mutational burden score, as estimated via next‐generation sequencing‐based gene panel testing, should be carefully interpreted as it could be affected by the DNA library concentration and sample type. Little is known about the preanalytical factors that could affect the tumor mutational burden score from next‐generation sequencing‐based multiplex gene panels, a promising biomarker to predict response to immune checkpoint inhibitors. This article investigates the association. [ABSTRACT FROM AUTHOR]

Abstract Adding undeclared species in meat-based food is an illicit practice with possible commercial, ethical and consumer health consequences. DNA-based quantitative methods are the most promising ones and their performance can be deeply affected by the quality of the input DNA. In this study, we performed a comprehensive evaluation of the DNA obtained using four common methodologies. DNA was isolated from three experimental food matrices (minced meat, ravioli filling and ragout) prepared with different percentages of beef and pork meat. Hundred and eighty DNA preps, including all matrix/percentage/kit combinations, were obtained and submitted to downstream quality assessment. Mean values and their statistical significance, obtained by parameter analysis and pairwise comparison, were used to calculate a DNA Quality Score (DQS), integrating quality parameters. DQS identified the salting-out based protocol as the best performing method for all the considered food matrices (p < 0.05); in DNA purification from ragout, the Cetyl-trimethylammonium bromide (CTAB) reagent-based protocol showed comparable results to those obtained by the salting-out method (p < 0.05). An optimal DNA purification is the first step for accurate species quantification in complex meat food. Highlights • Experimental meat-food matrices prepared in the lab. • DNA isolation based on different methodologies. • Comprehensive analysis of DNA quality from meat foods. • Development of a DNA Quality Score (DQS). [ABSTRACT FROM AUTHOR]

Solanecio biafrae (Oliv. & Hiern) C. Jеffrеу, an indigenous vegetable of the humid tropics, is rich in minerals and easy to cultivate, but its vegetative growth is slow, making production fall short of demand. An efficient and effective genomic DNA-extraction method is necessary for a comprehensive molecular analysis to increase leaf yield and further improve proximate and mineral contents of the vegetable. Cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), and Qiagen kit (QIAGEN) methods were evaluated for extraction of genomic DNA from young leaves suitable for PCR amplification for molecular analyses. Quantity and quality of the DNA were examined spectrophotometerically and with gel electrophoresis. Amplification of the DNA by PCR for molecular analysis was assessed with five inter-simple sequence repeat (ISSR) primers. The mean DNA yield using QIAGEN, CTAB, and SDS methods were 71.0, 35.4, and 38.8 µg∙µL−1∙g−1 of tissue, respectively. The mean 260/280 nm ratios of QIAGEN, CTAB, and SDS were 1.84, 1.83, and 1.79, respectively. Gel electrophoresis indicated presence of DNA free of contaminants in all samples using three methods. A total of 270 distinct bands were produced by five ISSR primers with similar patterns among three DNA-extraction methods, ranging from 200-2900 base pairs. Three methods are capable of extracting adequate genomic DNA suitable for PCR amplification. The QIAGEN method produced the highest DNA yield, which was of the same quality with DNA obtained using CTAB and SDS methods. [ABSTRACT FROM AUTHOR]

Despite the raising preoccupation, the critical question of how the plant community is composed belowground still remains unresolved, particularly for the conservation priority types of vegetation. The usefulness of metabarcoding analysis of the belowground parts of the plant community is subjected to a considerable bias, that often impedes detection of all species in a sample due to insufficient DNA quality or quantity. In the presented study we have attempted to find environmental factors that determine the amount and quality of DNA extracted from total plant tissue from above- and belowground samples (1000 and 10,000 cm2). We analyzed the influence of land use intensity, soil properties, species composition, and season on DNA extraction. The most important factors for DNA quality were vegetation type, soil conductometry (EC), and soil pH for the belowground samples. The species that significantly decreased the DNA quality were Calamagrostis epigejos, Coronilla varia, and Holcus lanatus. For the aboveground part of the vegetation, the season, management intensity, and certain species—with the most prominent being Centaurea rhenana and Cirsium canum—have the highest influence. Additionally, we found that sample size, soil granulation, MgO, organic C, K2O, and total soil N content are important for DNA extraction effectiveness. Both low EC and pH reduce significantly the yield and quality of DNA. Identifying the potential inhibitors of DNA isolation and predicting difficulties of sampling the vegetation plots for metabarcoding analysis will help to optimize the universal, low-cost multi-stage DNA extraction procedure in molecular ecology studies.

Introduction: Exploration of crime scene devices left over and its surrounding is of significance in forensic identification. The significance is due to its association and/or potential harbor of biological evidence; ie., blood, blood spots, semen patches, vaginal swabs, buccal swabs, bones, or trace evidence. Such harboring of evidence from humankind proposes traceability under Locard's principle of contact. Materials and Methods : Processing of cerumen expelled or traces logged from the 30 used earphones were subjected to 0, 1, 7, 14 and 20 days room temperature exposure. Results : Length of exposure has a significant effect on decreasing the level of DNA steadily. Visualization of the results of PCR on 143bp (HVS I, nt 16268-16410) mtDNA D-loop region showed a positive (+) detection only for day 1 (4 [66.67%] samples) and day 7 (3[50%] samples). Visualization of the results of PCR on the 126bp (HVS II, nt 34-159) mtDNA D-loop region showed a positive (+) detection only for day 1 (2 [33.37%] of samples) and day 7 (6 [100%] of samples). Conclusion: In conclusion, the length of exposure to room temperature has an effect on the quality of earphone swab-derived DNA. Decreased levels of earphone swab-derived DNA showed a significance value (p<0.005) with regard to the effect of room temperature exposure. [ABSTRACT FROM AUTHOR]

Aim of the study: One of the critical steps in molecular oncology diagnostics is obtaining high quality genomic DNA. Therefore, it is important to evaluate and compare the techniques used to extract DNA from tissue samples. Since formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for both retrospective and prospective studies, we compared three commercially available methods of nucleic acid extraction in terms of quantity and quality of isolated DNA. Material and methods: Slides prepared from 42 FFPE blocks were macro-dissected. Resulting material was divided and processed simultaneously using three extraction kits: QIAamp DNA FFPE Tissue Kit (QIAGEN), Cobas DNA Sample Preparation Kit (Roche Molecular Systems) and Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega). Subsequently, quantity and quality of obtained DNA samples were analysed spectrophotometrically (NanoDrop 2000, Thermo Scientific). Results of quantitative analysis were confirmed by a fluorometric procedure (Qubit 3.0 Fluorometer, Life Technologies). Results: The results demonstrated that the yields of total DNA extracted using either Maxwell or Cobas methods were significantly higher compared to the QIAamp method (p < 0.001). The Maxwell Extraction Kit delivered DNA samples of the highest quality (p < 0.01). However, the highest total yield of extracted DNA was achieved with the Cobas technique, which may be due to a higher volume of eluate compared to the Maxwell method. Conclusions: To our knowledge, this is the first paper which directly compares three extraction methods: Cobas, Maxwell and QIAamp. The data herein provide information required for the selection of a protocol that best suits the needs of the overall study design in terms of the quantity and quality of the extracted DNA. [ABSTRACT FROM AUTHOR]

The low efficiency of some nucleic acid extraction protocols and the high cost of commercial products, derives in the comparison between methods. In the present work three DNA extraction methods were compared from soybean, to obtain nucleic acids of adequate concentration and quality for PCR amplification. The protocols studied included the methods with 1% and 3% CTAB solutions, with 1% sarcosine and with phenol/chloroform. The experiments were carried out in the DNA and Genomics laboratory of the National Genetic Resources Center-INIFAP. The yield, purity, integrity and functionality of the obtained nucleic acids were evaluated. In all methods, adequate DNA yield was achieved, however, the required purity of the material was only obtained with the phenol/chloroform solution. With the methods of CTAB at 1% and 3% and sarcosine, PCR inhibiting substances were observed, while, with phenol/chloroform, the values of the A260/280 ratio were in a range of 1.96 to 2.00 and the A260/230 ratio in a range of 1.75 to 2.44, with significant differences (p< 0.0001) with the rest of the methods, in addition, the DNA was of high molecular weight and the rbcL gene was amplified by PCR in all the samples. The use of the phenol/chloroform protocol allowed to obtain from soybean, nucleic acids of adequate concentration and quality for PCR amplification. [ABSTRACT FROM AUTHOR]

Entomological collections are the target of various insect pests, e.g. carpet beetles (Dermestidae) and booklice (Psocoptera) which can damage and completely destroy dry specimens in a relatively short time. Collections in the National Museum, Czech Republic (NMP) including the entomological ones are protected by fumigation using commercially available smoke shells 'Cytrol Super SG'; fumigation is performed twice a year. The active insecticidal substance of these smoke shells is cypermethrin (6.25%). We tested whether the repeated cypermethrin fumigation of the NMP entomological collections negatively affects the quality of mitochondrial DNA in dry specimens and prevents the subsequent use of these samples for molecular analyses required for identification, taxonomy, systematics, and phylogenetic studies. We used 32 freshly fi xed specimens of the fl ower chafer Oxythyrea funesta (Poda von Neuhaus, 1761) and 32 freshly fi xed specimens of the brown-tailed cockroach Supella longipalpa (Fabricius, 1798). One half of specimens of both species was stored outside NMP and not fumigated (negative control), and the other half was deposited in collection hall with the NMP insect collection and directly exposed to the fumigation. Subsequently, all specimens were processed in a molecular laboratory under a standardized protocol using one leg as the source tissue after each fumigation, and the 658 bp long barcoding region of the cytochrome oxidase I (cox1) as the testing gene fragment. Results of the PCR product electrophoresis and the sequences acquired confirmed that the repeated fumigation had no negative effect on tested samples. [ABSTRACT FROM AUTHOR]

Freezing is a practical method for meat preservation but the quality of frozen meat can deteriorate with storage time. This research investigated the effect of frozen storage time (up to 66 months) on changes in DNA yield, purity and integrity in beef, and further analyzed the correlation between beef quality (moisture content, protein content, TVB-N value and pH value) and DNA quality in an attempt to establish a reliable, high-throughput method for meat quality control. Results showed that frozen storage time influenced the yield and integrity of DNA significantly (p < 0.05). The DNA yield decreased as frozen storage time increased due to DNA degradation. The half-life (t 1/2  = ln2/0.015) was calculated as 46 months. The DNA quality degraded dramatically with the increased storage time based on gel electrophoresis results. Polymerase chain reaction (PCR) products from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) were observed in all frozen beef samples. Using real-time PCR for quantitative assessment of DNA and meat quality revealed that correlations could be established successfully with mathematical models to evaluate frozen beef quality. [ABSTRACT FROM AUTHOR]

Long-term storage of whole blood can affect the integrity of DNA if it is not done under optimal conditions. The aim of this study was to determine whether long-term storage (2–19 years) of whole blood samples at −30°C had a negative effect on the quality or quantity of genomic DNA that could be recovered at extraction. Genomic DNA was isolated from 2758 whole blood samples collected in 4 mL EDTA vacutainers from 1997 to 2012. DNA was extracted using the Qiagen® FlexiGene® DNA kit. The average storage duration at −30°C was 12 years. The quality and quantity of the isolated DNA were assessed using spectrophotometry (NanoDrop™), a fluorometric assay for double-stranded DNA (Qubit™), and agarose gel electrophoresis. The mean DNA yield per sample was found to be 114 μg from whole blood volumes that ranged from 0.5 to 4 mL. The mean A260/280 ratio and median A260/280 ratios were both 1.8. No correlation was found between the duration of storage and the total yield or the quality of DNA extracted. These data suggest that high-quality DNA can be extracted from whole blood samples that are stored at −30°C for up to 19 years. [ABSTRACT FROM AUTHOR]

DNA extraction methods were evaluated for the yield and purity of DNA recovered from mycorrhized roots and whether the recovered DNA is suitable for amplification of arbuscular mycorrhizal (AM) fungal SSU rDNA. The DNeasy Plant Mini Kit and three extraction buffers were used alone or in combination with either polyvinylpyrrolidone (PVP), polyvinylpolypyrrolidone (PVPP) and/or activated charcoal (AC). Among the extraction methods tested, those based on the CTAB buffers yielded more DNA than those based on the TE buffer and the DNeasy Plant Mini Kit. Moreover, the use of AC alone or in combination with PVPP reduced DNA yield, while it significantly improved the purity of recovered DNA, whatever the extraction buffer. On the other hand, the success of nested-PCR amplification was negatively correlated with the amount of template DNA and positively correlated with the purity of recovered DNA. Three methods based on the TE buffer, two on the CTAB-βM buffer and one on the DNeasy Plant Mini Kit produced high-quality DNA in terms of purity and PCR performance. However, the TE buffer-based methods are less time consuming than the CTAB-βM buffer-based methods, and cheaper than the method based on the DNeasy Plant Mini Kit.

Responses to milk sterilization are usually evaluated only in terms of physicochemical properties and microbial safety, thus undervaluing the importance of DNA quality in an authentication process by methods based on PCR. Because DNA is a heat-sensitive molecule, we hypothesized that the heating process may impair the detection or quantification of DNA in raw fresh milk (FM) or reconstituted milk (RM), and that differences in DNA quality might exist between FM and RM. We thus investigated the effects of sterilization on the quality of DNA extracted from FM or RM; differences in DNA quality between FM and RM were also evaluated. The quality of extracted DNA from FM or RM was assessed by the specific detection of FM or RM composition in goat milk mixtures using primers targeting the bovine 12S gene, as well as by monitoring DNA yield, purity, ratio of mitochondrial (mt) to nuclear (n) DNA, and the level of DNA degradation. Polymerase chain reaction readily detected both untreated and heat-treated FM or RM in cow-goat milk mixtures, and gave a good sensitivity threshold (0.1%) under all sterilization conditions. The DNA yield and mtDNA:nDNA ratio of FM and RM varied significantly during the sterilization process. These results demonstrated that the sterilization altered the quantification of DNA in FM or RM during sterilization, but DNA could still be readily detected in sterilized FM or RM by PCR. Furthermore, we noted significant differences in DNA integrity, yield, and mtDNA:nDNA ratio between FM and RM during sterilization, which may have potential as a means to distinguish FM and RM. [ABSTRACT FROM AUTHOR]

Background: Genomic DNA extracted from species of Cactaceae is often contaminated with significant amounts of mucilage and pectin. Pectin is one of the main components of cellular walls, whereas mucilage is a complex polysaccharide with a ramified structure. Thus, pectin- and mucilage-free extraction of DNA is a key step for further downstream PCR-based analyses. Results: We tested our DNA extraction method on cladode tissue (juvenile, adult, and herbaria exemplars) of 17 species of Opuntia Mill., which are characterized by a large quantity of pectin and mucilage. Conclusion: We developed a method for the extraction of gDNA free of inhibitory compounds common in species of Opuntia Mill., such as pectin and mucilage. Compared to previously extraction protocols, our method produced higher yields of high-quality genomic DNA. [ABSTRACT FROM AUTHOR]

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