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

The measurement and analysis of snail biometrics help to identify and differentiate species, study their growth, and understand their ecological relationships. The growth pattern, DNA yield and purity of Bulinus. globosus from River Wudil, Kano State, were investigated using standard methods. The mean ranges in mm and g from 7.9±2.9, 5.7±2.5, 4.8±1.8, 3.4±1.2, 1.5±0.2, 1.6±0.0, 2.4±0.2, 1.2±0.1, 1.4±0.1, 65.8±1.9 and 22.2±0.4 were recorded for length, width, aperture height, aperture width, height width ratio, height and aperture height ratio, height and aperture width ratio, width and aperture height ratio, aperture height and aperture width ratio, shell weight and flesh weight respectively. About 33.7 % of weight of the snail is made up of flesh by weight. The regression coefficient "b" showed that B. globosus exhibited a negative allometric growth pattern while the correlation coefficient (r) in all the shell parameters were below 1, indicating a weak correlation between the parameters. Mean DNA yield and purity were 120.03 ± 5.10 ng/µl and 1.81 ± 1.21. It is concluded that the freshwater snail, B. globusus from River Wudil exhibits negative allometric growth pattern and its DNA yield is well above the minimum standard (A260/A280 ratio of 1.7-2.0) while the pure extracts are good enough for further molecular study. [ABSTRACT FROM AUTHOR]

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.

ABSTRACT PCR can be a supplement to Treponema serological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield of Treponema pallidum subsp. pallidum DNA extraction from blood. We developed and verified the efficacy of a quantitative PCR (qPCR) assay that utilizes TaqMan technology to specifically detect T. pallidum DNA by targeting the polA gene. Simulation media with 106 to 100 treponemes/mL were prepared in normal saline (NS), whole blood, plasma, and serum, and RBC lysis pretreatment was performed on a portion of whole blood. Then, blood samples drawn from 50 syphilitic rabbits were divided in parallel into five groups, labeled whole blood, whole blood/lysed RBCs, plasma, serum, and blood cells/lysed RBCs. DNA extraction and qPCR detection were performed. The detection rate and copy number were compared among different groups. The polA assay showed good linearity and an excellent amplification efficiency of 102%. In the simulated blood samples, the detection limit of the polA assay reached 1 × 102 treponemes/mL in whole blood/lysed RBCs, plasma, and serum. However, the detection limit was only 1 × 104 treponemes/mL in NS and whole blood. Among the blood samples from syphilitic rabbits, whole blood/lysed RBCs showed the best detection rate (82.0%), while the detection rate for whole blood was only 6%. The copy number of whole blood/lysed RBCs was higher than that of whole blood. RBC lysis pretreatment can significantly improve the yield of T. pallidum DNA from whole blood, and the yield is better than that from whole blood, plasma, serum, and blood cells/lysed RBCs. IMPORTANCE Syphilis is a sexually transmitted disease caused by T. pallidum that can spread into the blood. T. pallidum DNA can be detected in blood by PCR but with low sensitivity. Few studies have applied RBC lysis pretreatment to blood T. pallidum DNA extraction. This study shows that the detection limit, detection rate, and copy number of whole blood/lysed RBCs were better than those of whole blood, plasma, and serum. After RBC lysis pretreatment, the yield of low concentrations of T. pallidum DNA was improved, and the low sensitivity of blood-based T. pallidum PCR was improved. Therefore, whole blood/lysed RBCs are the ideal sample for acquiring blood T. pallidum DNA.

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]

• Significantly higher PSA test positivity rate for ForensiX SafeDry at T 48. • Significantly increased DNA yield in Sarstedt XL samples compared to ForensiX Kit. • Mostly complete autosomal male DNA profiles from sperm cell fractions over all time points. • Potential malfunction of Sarstedt XL drying membrane can lead to microbial growth. This study compared the currently used swab Prionics ForensiX Evidence Collection Kit with the alternatives Prionics ForensiX Evidence Collection Tube SafeDry and Sarstedt Forensic Swab XL. Volunteers provided intravaginal swabs collected with all swab types at specific time points after unprotected sexual intercourse. Quantifiable DNA, detectability of seminal fluid component (prostate specific antigen, PSA) and spermatozoa were evaluated to find the best-performing swab type. While Sarstedt XL showed significantly higher DNA quantities for sperm cell fractions than ForensiX Kit , the more concise PSA test results clearly favour ForensiX SafeDry. Reassuringly, mostly complete autosomal STR profiles of male components were obtained for sperm cell fractions at all time points and tested swabs. Switching to the higher performing ForensiX SafeDry with improved sampling and processing properties will also benefit victims, medical personnel, and investigators. [ABSTRACT FROM AUTHOR]

• Blood recovered from snow can yield viable DNA profiles suitable for court. • Various substrates commonly used by Forensic Identification Officers are sufficient mediums for recovering high yield blood from snow, where replicates performed consistently. • Slight variability in DNA yields and genotyping performance across the three substrates was observed, attributable to differences in methodology/evidence collection. Successful DNA typing of forensically relevant evidence is reliant on both the quality and quantity of biological material recovered from a crime scene. In geographical areas of the world exposed to cold climates, it is not uncommon for biological evidence to encounter a diversity of challenging surfaces and environments, including snowy surfaces. Currently, there is no standard protocol for recovery of bloodstain evidence in snow and very few publications exploring adequate methods of recovering biological evidence from snowy surfaces. In this study, three common substrates (e.g., cotton swabs, FTA paper, and untreated filter paper) utilized by investigators for evidence recovery were evaluated for their ability to recover human blood (DNA) evidence from snow that would be viable for traditional forensic DNA typing. Each biological sample was extracted and quantified to evaluate the quality and quantity of DNA recovered. All samples yielded sufficient non-degraded DNA to proceed with DNA profiling, where complete DNA profiles were generated from each collection substrate. The experimental findings presented herein demonstrate that the ability to recover viable DNA from human blood collected on surface snow is possible using all three collection methods tested. [ABSTRACT FROM AUTHOR]

A swab is a tool for obtaining buccal DNA from buccal mucus for biological analysis. The acquisition of a sufficient amount and high quality of DNA is an important factor in determining the accuracy of a diagnosis. A microneedle swab (MN swab) was developed to obtain more oral mucosal tissues non-invasively. Eight types of MN swabs were prepared with varying combinations of patterns (zigzag or straight), number of MNs, intervals of MNs, and sharpness of tips. When MN swab was applied up to 10 times, the tissue amount and DNA yield increased compared to commercial swabs. A zigzag pattern of microneedles was found to be more efficient than a straight pattern and increasing the number of microneedles in an array increased the DNA yield. The MN swab collected about twice the DNA compared to the commercial swab. In an in vivo test using mini pigs, the lower cycle threshold values of mucosal samples collected with MN swabs compared to samples collected with commercial swabs indicated that a greater amount of DNA was collected for SNP genotyping. A polymer MN swab is easy to manufacture by a single molding process, and it has a greater sampling capacity than existing commercial swabs.

DNA sampling and typing are used for identifying missing persons or war victims. In recent forensic studies, little focus has been placed on determining intra-bone variability within a single skeletal element. When dealing with aged human bones, complete skeletal remains are rarely present. In cases in which only the torso is available, studies have shown that ribs are one of the most appropriate samples, but intra-bone variability has not yet been studied. A higher degree of remodeling was found to contribute to higher DNA yield in the parts of the skeletal element where the most strain is concentrated. This study explores intra-bone variability in proximal, middle, and distal parts of the first human rib by determining the quantity and quality of DNA using the PowerQuant System (Promega) and autosomal STR typing success using the PowerPlex ESI 17 Fast System (Promega). Thirty first ribs from a single Second World War mass grave were sampled. No variation in DNA degradation was observed across the individual rib. The highest quantity of DNA was measured in the proximal part of the first rib, and in all ribs except three, full or almost full genetic profiles were obtained. Thus, when only the torso is present in archaeological or medico-legal cases, first ribs are recommended to be collected if possible, and the proximal or vertebral ends should be sampled for genetic analysis. [ABSTRACT FROM AUTHOR]

The genus Tamarix consists of about 54 species that mainly grow in saline areas of deserts and semi-deserts. This genus is chemically characterized by the presence of tannins, flavonoids, anthocyanins and essential oils which interfere with the extraction of pure genomic DNA. Thus it is necessary to optimize extraction protocols to minimize the influence of these compounds to the lowest level. The present study compares the efficiency of five different approaches to extract total genomic DNA in Tamarix species, showing significant differences in the extracted DNA contents and quality,by using Kit (DNP TM Kit), CTAB DNA extraction method by Murray and Thompson, Sahu et al., Nalini et al. and Bi et al., for the extraction of DNA from Tamarix species. Our results showed significant differences in DNA contents between these five methods. The quantity and quality of extracted genomic DNA were checked by the spectrophotometer, Nano-Drop and and agarose gel electrophoresis analysis. Finally, a PCR-based method was also applied to verify the amplification efficiency for two molecular markers (ITS and ISSR).. In the present study, the genetic diversity of 96 Tamarix individuals species and 8 populations were studied using 10 ISSR markerswhile for nrDNA ITS 8 species samples were used. The method of Nalini et al., provided best results (207 ng/μL) in terms of quantity and quality ofDNA. Our results proposed that this method could be effective for plants with the same polysaccharides, proteins and polyphenols components. The advantage of this method is simple and fast as it does not involve time consuming steps such as incubation at higher temperatures, and also do not requires expensive chemicals such as proteinase K, liquid nitrogen. ,. The success of this method in obtaining high-quality genomic DNA has been demonstrated in the Tamarix species group and the reliability of this method has been discussed.

Sediment is thought to be a vital reservoir for antibiotic resistance genes (ARGs). Often, studies describing and comparing ARGs and their potential hosts in sediment are based on single DNA extractions. To date, however, no study has been conducted to assess the influence of DNA extraction efficiency on ARGs in sediment. To determine whether the abundance of ARGs is underestimated, we performed five successive extraction cycles with a widely used commercial kit in 10 sediment samples collected from the Haihe River and Bohai Bay. Our results showed that accumulated DNA yields after five extractions were 1.8–3.1 times higher than that by single DNA extractions. High-throughput sequencing showed that insufficient DNA extraction could generate PCR bias and skew community structure characterization in sediment. The relative abundances of some pathogenic bacteria, such as Enterobacteriales, Lactobacillales, and Streptomycetales, were significantly different between single and successive DNA extraction samples. In addition, real-time fluorescent quantitative PCR (qPCR) showed that ARGs, intI 1, and 16S rRNA gene abundance strongly increased with increasing extraction cycles. Among the measured ARGs, sulfonamide resistance genes and multidrug resistance genes were dominant subtypes in the study region. Nevertheless, different subtypes of ARGs did not respond equally to the additional extraction cycles; some continued to have linear growth trends, and some tended to level off. Additionally, more correlations between ARGs and bacterial communities were observed in the successive DNA extraction samples than in the single DNA extraction samples. It is suggested that 3–4 additional extraction cycles are required in future studies when extracting DNA from sediment samples. Taken together, our results highlight that performing successive DNA extractions on sediment samples optimizes the extractable DNA yield and can lead to a better picture of the abundance of ARGs and their potential hosts in sediments. [ABSTRACT FROM AUTHOR]

DNA analysis of Second World War skeletal remains is challenging because of the limited yield of DNA that is usually recovered. Recent forensic research has focused on determining which skeletal elements are superior in their preservation of DNA, and little focus has been placed on measuring intra-bone variability. Metatarsals and metacarpals outperformed all the other bones in DNA yield when analyzing all representative skeletal elements of three Second World War victims, and intra-bone variability was not studied. Soft-tissue remnants were found to contribute to higher DNA yield in trabecular bone tissue. Because metatarsals and metacarpals are composed of trabecular epiphyses and a dense diaphysis, the goal of this study was to explore intra-bone variability in DNA content by measuring nuclear DNA quantity and quality using the PowerQuant System (Promega). A total of 193 bones from a single Second World War mass grave were examined. From each bone, DNA was extracted from the compact diaphysis and from both spongy epiphyses combined. This study confirms higher DNA quantity in epiphyses than diaphyses among all the bones analyzed, and more DNA was obtained from metacarpal epiphyses than from metatarsal epiphyses. Therefore, whenever the possibility for sampling both metacarpals and metatarsals from skeletal remains exists, collecting metacarpals is recommended. In cases in which the hands are missing, metatarsals should be sampled. In any case, epiphyses are a richer source of DNA than diaphyses. [ABSTRACT FROM AUTHOR]

Sediment is thought to be a vital reservoir for antibiotic resistance genes (ARGs). Often, studies describing and comparing ARGs and their potential hosts in sediment are based on single DNA extractions. To date, however, no study has been conducted to assess the influence of DNA extraction efficiency on ARGs in sediment. To determine whether the abundance of ARGs is underestimated, we performed five successive extraction cycles with a widely used commercial kit in 10 sediment samples collected from the Haihe River and Bohai Bay. Our results showed that accumulated DNA yields after five extractions were 1.8–3.1 times higher than that by single DNA extractions. High-throughput sequencing showed that insufficient DNA extraction could generate PCR bias and skew community structure characterization in sediment. The relative abundances of some pathogenic bacteria, such as Enterobacteriales, Lactobacillales, and Streptomycetales, were significantly different between single and successive DNA extraction samples. In addition, real-time fluorescent quantitative PCR (qPCR) showed that ARGs, intI1, and 16S rRNA gene abundance strongly increased with increasing extraction cycles. Among the measured ARGs, sulfonamide resistance genes and multidrug resistance genes were dominant subtypes in the study region. Nevertheless, different subtypes of ARGs did not respond equally to the additional extraction cycles; some continued to have linear growth trends, and some tended to level off. Additionally, more correlations between ARGs and bacterial communities were observed in the successive DNA extraction samples than in the single DNA extraction samples. It is suggested that 3–4 additional extraction cycles are required in future studies when extracting DNA from sediment samples. Taken together, our results highlight that performing successive DNA extractions on sediment samples optimizes the extractable DNA yield and can lead to a better picture of the abundance of ARGs and their potential hosts in sediments.

Phylogenetic datasets are now commonly generated using short-read sequencing technologies unhampered by degraded DNA, such as that often extracted from herbarium specimens. The compatibility of these methods with herbarium specimens has precipitated an increase in broad sampling of herbarium specimens for inclusion in phylogenetic studies. Understanding which sample characteristics are predictive of sequencing success can guide researchers in the selection of tissues and specimens most likely to yield good results. Multiple recent studies have considered the relationship between sample characteristics and DNA yield and sequence capture success. Here we report an analysis of the relationship between sample characteristics and sequencing success for nearly 8,000 herbarium specimens. This study, the largest of its kind, is also the first to include a measure of specimen quality (“greenness”) as a predictor of DNA sequencing success. We found that taxonomic group and source herbarium are strong predictors of both DNA yield and sequencing success and that the most important specimen characteristics for predicting success differ for DNA yield and sequencing: greenness was the strongest predictor of DNA yield, and age was the strongest predictor of proportion-on-target reads recovered. Surprisingly, the relationship between age and proportion-on-target reads is the inverse of expectations; older specimens performed slightly better in our capture-based protocols. We also found that DNA yield itself is not a strong predictor of sequencing success. Most literature on DNA sequencing from herbarium specimens considers specimen selection for optimal DNA extraction success, which we find to be an inappropriate metric for predicting success using next-generation sequencing technologies.

Phylogenetic datasets are now commonly generated using short-read sequencing technologies unhampered by degraded DNA, such as that often extracted from herbarium specimens. The compatibility of these methods with herbarium specimens has precipitated an increase in broad sampling of herbarium specimens for inclusion in phylogenetic studies. Understanding which sample characteristics are predictive of sequencing success can guide researchers in the selection of tissues and specimens most likely to yield good results. Multiple recent studies have considered the relationship between sample characteristics and DNA yield and sequence capture success. Here we report an analysis of the relationship between sample characteristics and sequencing success for nearly 8,000 herbarium specimens. This study, the largest of its kind, is also the first to include a measure of specimen quality ("greenness") as a predictor of DNA sequencing success. We found that taxonomic group and source herbarium are strong predictors of both DNA yield and sequencing success and that the most important specimen characteristics for predicting success differ for DNA yield and sequencing: greenness was the strongest predictor of DNA yield, and age was the strongest predictor of proportion-on-target reads recovered. Surprisingly, the relationship between age and proportion-on-target reads is the inverse of expectations; older specimens performed slightly better in our capture-based protocols. We also found that DNA yield itself is not a strong predictor of sequencing success. Most literature on DNA sequencing from herbarium specimens considers specimen selection for optimal DNA extraction success, which we find to be an inappropriate metric for predicting success using next-generation sequencing technologies. [ABSTRACT FROM AUTHOR]

Summary: Robust and accurate identification of root-lesion nematodes (Pratylenchus spp.) is an essential step for determining their potential threat to crop yields and, consequently, development of an efficient agronomic management strategy. It is recognised that DNA-based techniques provide rapid identification of a range of plant-parasitic nematodes including Pratylenchus spp. Efficient and repeatable DNA extraction is central to molecular methodologies. Here, six common DNA extraction protocols were compared to evaluate their efficiency to obtain quality DNA samples for Pratylenchus penetrans. Samples with five and ten individuals of P. penetrans were successfully extracted and amplified by all extraction methods tested, whereas samples with a single nematode presented challenges for DNA amplification. Among all methods tested, the DNA extraction protocol with glass beads proved to be efficient for P. penetrans and all other species tested (P. crenatus , P. neglectus and P. thornei), generating high quality DNA at comparatively low cost and with a rapid sample throughput. [ABSTRACT FROM AUTHOR]

Biliary tract cancer (BTC) is a rare malignancy with a long disease course and an overall poor prognosis. Despite multiple chemotherapy agents, there is no defined second-line treatment opportunity for advanced BTCs. In the era of precision oncology, NGS plays an important role in identifying mutations that may predict the molecular pathomechanism and manage the BTC therapy. The peripheral blood liquid biopsy (LB) of cancer patients represents variable amounts of cell-free DNA (cfDNA) released from tumor foci of any anatomical location. Our study aimed to identify somatic mutations and tumor variant burden (TVB) in cell-free and matched tumor DNA. We found a positive correlation between the estimated tumor volume and cfDNA yield (r = 0.9326, p < 0.0001). Comparing tissue and LB results, similar TVB was observed. SNVs were proven in 84% of the cases, while in two cases, only the LB sample was informative for molecular analysis. The most important aberrations in BTCs, such as FGFR2, IDH1, IDH2, KRAS, and TP53, could be detected in matched LB samples. Our prospective study demonstrates a minimally invasive testing approach to identify molecular genetic alterations in cholangiocarcinoma and gallbladder cancers. Clinical applications of cfDNA reflect by capturing the outstanding spatial tumor heterogeneity and guarantee novel aspects for the precision oncology treatment.

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

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]

Cigarette butts found in crime scenes may be used to identify persons and link them to a crime through DNA prof iling of epithelial cells from saliva stains on these materials. Downstream analysis of cigarette butts poses some challenges because these are often exposed to chemical contaminants and environmental conditions which lead to DNA degradation. In this study, several factors were tested to compare the amount and quality of DNA obtained from cigarette butts extracted using an organic procedure and the QIAamp® DNA Micro Kit (QIAGEN). Results show that exposure to an outside environment had a significant effect on DNA yield and amplif i ability for both extraction procedures. Prolonged storage of cigarette butts of up to six months affected the amount of DNA that can be extracted using the QIAamp® DNA Micro Kit. However, complete DNA prof iles can be generated from cigarette butts stored for six months provided that these samples are stored indoors under controlled temperature conditions and with minimal exposure to contaminants. [ABSTRACT FROM AUTHOR]

Background: Next generation sequencing (NGS) to detect actionable genetic abnormalities is standard of care in advanced stage lung adenocarcinoma. Many studies have shown that the molecular results obtained from fine needle aspiration cytology material are comparable to those obtained from formalin‐fixed tissue samples. We undertook this study to validate DNA extraction from cytology material for molecular studies and to find any correlation between DNA yield, pattern of tumour cells and tumour fraction. Methods: DNA was extracted from 34 cytology slides of pulmonary adenocarcinoma cases with predetermined EGFR mutation status. Cytology slides were reviewed for pattern of tumour distribution and tumour fraction. NGS was performed on five slides with variable DNA and compared with original results. Results: There were 14 alcohol‐fixed and 20 air‐dried smears. The mean DNA yield was 1.74 μg and median of 0.4 μg (range, 0.02‐21 μg). Tumour fractions varied from 10% to 90%. No correlation was found between tumour fraction and DNA yield (P = 0.14). The mean DNA yield was high in slides with tumour throughout the slide (sheets or scattered clusters) as compared to rare scattered clusters and/or single cells. EGFR mutation was found in four of the five cases sent for NGS lung panel while one case revealed BRAF mutation. Conclusions: DNA with good quantity and quality can be extracted from the cytology slides for NGS irrespective of type of fixation. DNA yield has better correlation with distribution pattern of tumour cells on the slides rather than tumour fraction. This paper describes the characteristics of cytology smears in neoplastic diseases which would be helpful in choosing the cytology smear to extract good quantity DNA for successful next generation sequencing which could impact the management of the patients. [ABSTRACT FROM AUTHOR]

Current sampling strategy for laboratories typing bones for human identification include samples obtained from femur, tooth and temporal bone. Latest studies suggest that the small bones of the hands and feet were very similar or even better in DNA yield. These bones can be easily sampled with a disposable scalpel and thus reduce potential DNA contamination. The aim of our study was to determine the suitability of metatarsals, metacarpals and phalanges for genetic identification. 48 bone samples from 8 different skeletons (six from 18th century and two from 3rd century) were obtained from 5 archaeological sites in Slovenia. In each skeleton, 6 different skeletal elements were sampled (temporal bone, molar, femur, metacarpal bone, metatarsal bone and proximal phalanx of the hand), and strict precautions followed to prevent contamination. Half of gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen), DNA content determined with the PowerQuant kit (Promega), and autosomal STR typing performed with the Investigator ESSplex Plus kit (Qiagen). Up to 8.75 ng DNA/g of powder was obtained from samples analyzed. The highest yields were detected in temporal bone and the lowest in femur. The success rate of STR typing was evaluated according to the number of successfully typed loci and a strong correlation between the success rate of STR typing and the amount of extracted DNA was confirmed. For all eight skeletons full consensus genetic profiles were determined from skeletal elements analyzed. Our findings suggest it would be suitable to include metatarsal and metacarpal bones in sampling strategy for human identification although further research is needed to substantiate the findings of this study. [ABSTRACT FROM AUTHOR]

Sample storage is of paramount importance in forensic genetics laboratories since only optimal storage enables successful recovery of DNA from old bones that contain very low amount of severely degraded DNA. When identification of missing persons from skeletal remains is completed, bone sample is routinely stored at -20 °C for long-term storage for retesting in future, if necessary. After molecular genetic analyses of Slovenian Second World War (WWII) victims, small fragments of femurs were stored at -20 °C. Reduction in DNA recovery has been observed in frozen liquid DNA extracts by some authors and the goal of our study was to explore how freezing of bone samples affects the preservation of DNA. To achieve this goal, the difference in DNA yield in extracts obtained from WWII bones analyzed in 2009 (data from published paper) and DNA yield in extracts obtained from the same bones (piece sampled next to the one used in 2009) taken out of the freezer after long-term storage on -20 °C for 10 years was examined, using the same extraction method and the same quantification kit. Up to 100 ng DNA/g of bone powder was obtained from 57 WWII femurs and up to 31 ng DNA/g of bone powder from the same femurs investigated after long-term storage in this study. 0,5 g of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 device (Qiagen) and DNA quantity determined with the Human Quantifiler kit (TFS). Statistical analysis showed significant difference in DNA yield in extracts obtained from WWII bones in 2009 and extracts obtained from the same bones stored at -20 °C after 10 years. As reported for frozen liquid DNA extracts, reduction in recovery of DNA was confirmed for frozen bone samples as well. [ABSTRACT FROM AUTHOR]

The Argentine Forensic Anthropology Team (EAAF) investigates human rights violations which took place in Argentina during the military government that ruled out from 1976 to 1983 and also cooperates in other countries with humanitarian crisis as well. Most of the bone remains that the EAAF analyses show long post mortem interval. Consequently, the skeletal remains may have been exposed to different conditions such as moisture, temperature and pH conditions, microorganisms and different types of soil that can produce various degrees of preservation. On the other hand, severely burnt remains can make the genetic analysis more complex. All the above mentioned influences the DNA quantity and quality. In the present study, we scored the samples to investigate the correlation between the bone macroscopic aspect and the concentration of the DNA extracted from it. Samples were classified into four major groups: score 1 was assigned to samples with very good preservation, score 2 to those showing slight superficial alterations. Score 3 and 4 samples showed regular and bad macroscopic appearance, respectively. Our results clearly supports that samples with good preservation show high well-preserved DNA concentrations and also a higher number of reportable STR markers in comparison to those ranked in the worst categories. This work proposes a useful guide that can help anthropologists to make a more efficient selection of aged-samples for genetic studies. [ABSTRACT FROM AUTHOR]

In this work, we employed real-time PCR analysis targeting tp0574 to investigate the effects of different processing procedures on the yield of T. pallidum DNA from blood to improve assay sensitivity. The T. pallidum DNA yields following red blood cell lysis pretreatment were 40.4 times greater from whole blood and 32.4 times greater from residual hematocytes than yields without pretreatment. For the simulated whole-blood experiments, the T. pallidum DNA yields from the lower layer were 2.8, 4.6, 7.3, 12.6, 15.24, 16.7, 65.1 and 73.1 times those from the upper layer following centrifugation at 500×, 1000×, 2000×, 4000×, 5000×, 7000×, 10,000× and 20,000 ×  g , respectively. However, the T. pallidum DNA yields from blood clots were only 1.0% at different centrifugal forces. The experiment with infected rabbit blood showed results similar to those mentioned above. In addition, sample processing time (within 48 h) and storage temperature (4 °C and 25 °C) did not affect T. pallidum DNA extraction efficiency. The T. pallidum DNA yield can be significantly improved by red blood cell lysis pretreatment and appropriate centrifugation. Furthermore, the T. pallidum DNA extraction yield is greater from whole blood or residual hematocytes from anti-coagulated blood than from plasma, serum or blood clots. [ABSTRACT FROM AUTHOR]

Skin swabbing, a minimally invasive DNA sampling method recently proposed for adult amphibians, was tested on the dendrobatid frog Allobates femoralis. I compared DNA yield from skin swabs and toe clips by evaluating obtained DNA concentrations and purity of extracts, as well as amplification success using eleven polymorphic microsatellite loci. I also tested whether storing skin swabs for two months at −20°C affected the properties of the extract or microsatellite analysis. Results show that skin swabs of adult A. femoralis suffered from high contamination and yielded significantly lower DNA quality and quantity, resulting in insufficient genotyping success, than DNA obtained from toe clips. The relatively dry skin in dendrobatid frogs may have impeded the collection of sufficient viable cells, and the presence of skin alkaloids and microbiota in the frog mucus may lead to high contamination load of skin swabs. [ABSTRACT FROM AUTHOR]

DNA extraction for molecular biological applications usually requires target optimized extraction procedures depending on the origin of the samples. For environmental samples, a range of different procedures has been developed. We compared the applicability and efficiency of ten selected DNA extraction methods published in recent literature using four different environmental samples namely: activated sludge from a domestic wastewater treatment plant, river sediment, anaerobic digestion sludge and nitrifying enrichment culture. We assessed the suitability of the extraction procedures based on both DNA yield and quality. DNA quantification was performed by both ultra violet (UV) spectrophotometry and fluorescence spectrophotometry after staining with PicoGreen. In our study, DNA yields based on UV measurement were overestimated in most cases while DNA yields from fluorescence measurements correlated well with the sample load on agarose gels of crude DNA. The quality of the DNA extracts was determined by gel electrophoresis of crude DNA and PCR products from 16S rDNA with the universal primer set 27f/1525r. It was observed that gel electrophoresis of crude DNA was not always suitable to evaluate DNA integrity and purity since interfering background substances (e.g. humic substances) were not visible. Therefore, we strongly recommend examining the DNA quality of both crude DNA and 16S rDNA PCR products by gel electrophoresis when a new extraction method is established. Summarizing, we found four out of ten extraction procedures being applicable to all tested samples without noticeable restrictions. The procedure G (according to the standard method 432_10401 of the Lower Saxony State Office for Consumer Protection and Food Safety) had the broadest application range over procedure J (published by Wilson, 2001). These were followed by procedures F (Singka et al., 2012) and A (Bourrain et al., 1999). All four extraction procedures delivered reliable and reproducible crude DNA and PCR products. From an economical point of view, all procedures tested during this study were cheaper compared to commercial DNA extraction kits. [ABSTRACT FROM AUTHOR]

Background: The current study was conducted to compare DNA yield, including normalization to nuclear area, DNA amplification functionality, and detection of KRAS mutations between matched fine-needle aspiration (FNA) specimens and pancreatic resections diagnostic of pancreatic ductal adenocarcinoma.Methods: A retrospective sample of 30 matched single FNA smears and macrodissected formalin-fixed, paraffin-embedded (FFPE) curls (2 5-μm curls) were compared by measuring the following: nuclear area (via digital image analysis), DNA yield (via NanoDrop spectrophotometry and Quantus fluorometry), and polymerase chain reaction threshold cycles for KRAS amplifications. Variants in KRAS codons 12/13 and 61 were detected by fluorescent melt curve analyses, followed by Sanger DNA sequencing.Results: Despite a similar nuclear area, FNA smears yielded greater DNA per nuclear area via 2 DNA quantification methods. KRAS codon 12 mutations were detected in 23 of 30 FNA specimens (77%) compared with 17 of 30 matched FFPE specimens (57%), for a concordance rate of 74%. No KRAS codon 13 or 61 mutations were detected.Conclusions: FNA specimens are a more optimal source of DNA, and represent an important resource in the preresection and postresection molecular analysis of pancreatic ductal adenocarcinoma. Cancer Cytopathol 2017;125:838-47. © 2017 American Cancer Society. [ABSTRACT FROM AUTHOR]

Wet-vacuum collection of forensic biological material has been shown to recover greater total DNA yields compared to traditional methods, such as wet swabbing. The Pulse Lavage System (PLS), an orthopedic surgical instrument, was evaluated in comparison to a forensic wet-vacuum device for the DNA collection and recovery of diluted bloodstains from seven substrates of varying porosity. Three different PLS models were evaluated, and each model yielded DNA concentrations which were comparable to the forensic wet-vacuum system, recovering 79–99 % relative to the wet-vacuum, which were overall not statistically different. Our results suggest that the PLS, though intended for medical use, has the potential to serve as an affordable alternative to the forensic wet-vacuum system. However, additional evaluation and modification to the PLS collection method may be warranted for complete optimization. • Wet-vacuum collection of biological material can recover more DNA than swabbing. • Despite the wet-vacuum's advantages, the upfront cost may hinder its widespread use. • Diluted blood was successfully collected with three different Pulse Lavage Systems. • Collection efficiencies of the Pulse Lavage Systems and wet-vacuum were comparable. [ABSTRACT FROM AUTHOR]

The extraction of DNA is a critical step for species identification by PCR analysis in processed food and feed products. In this study, eight DNA extraction procedures were compared—DNeasy Blood and Tissue Kit, DNeasy mericon Food Kit, chemagic DNA Tissue 10 Kit, Food DNA Isolation Kit, UltraPrep Genomic DNA Food Mini Prep Kit, High Pure PCR Template Preparation Kit, phenol—chloroform extraction, and NucleoSpin Food—Using self-prepared samples from both raw and heat-processed and/or mechanically treated muscles and different types of meat products and pet food (pork, beef, and chicken). The yield, purity, and suitability of DNA for PCR amplification was evaluated. Additionally, comparisons between the effectiveness of various extraction methods were made with regard to price, and labor- and time-intensiveness. It was found that the DNeasy mericon Food Kit was the optimal choice for the extraction of DNA from raw muscle, heat-treated muscle, and homemade meat products from multiple and single species.

PCR can be a supplement to Treponema serological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield of Treponema pallidum subsp. pallidum DNA extraction from blood. We developed and verified the efficacy of a quantitative PCR (qPCR) assay that utilizes TaqMan technology to specifically detect T. pallidum DNA by targeting the polA gene. Simulation media with 10 6 to 10 0 treponemes/mL were prepared in normal saline (NS), whole blood, plasma, and serum, and RBC lysis pretreatment was performed on a portion of whole blood. Then, blood samples drawn from 50 syphilitic rabbits were divided in parallel into five groups, labeled whole blood, whole blood/lysed RBCs, plasma, serum, and blood cells/lysed RBCs. DNA extraction and qPCR detection were performed. The detection rate and copy number were compared among different groups. The polA assay showed good linearity and an excellent amplification efficiency of 102%. In the simulated blood samples, the detection limit of the polA assay reached 1 × 10 2 treponemes/mL in whole blood/lysed RBCs, plasma, and serum. However, the detection limit was only 1 × 10 4 treponemes/mL in NS and whole blood. Among the blood samples from syphilitic rabbits, whole blood/lysed RBCs showed the best detection rate (82.0%), while the detection rate for whole blood was only 6%. The copy number of whole blood/lysed RBCs was higher than that of whole blood. RBC lysis pretreatment can significantly improve the yield of T. pallidum DNA from whole blood, and the yield is better than that from whole blood, plasma, serum, and blood cells/lysed RBCs. IMPORTANCE Syphilis is a sexually transmitted disease caused by T. pallidum that can spread into the blood. T. pallidum DNA can be detected in blood by PCR but with low sensitivity. Few studies have applied RBC lysis pretreatment to blood T. pallidum DNA extraction. This study shows that the detection limit, detection rate, and copy number of whole blood/lysed RBCs were better than those of whole blood, plasma, and serum. After RBC lysis pretreatment, the yield of low concentrations of T. pallidum DNA was improved, and the low sensitivity of blood-based T. pallidum PCR was improved. Therefore, whole blood/lysed RBCs are the ideal sample for acquiring blood T. pallidum DNA., Competing Interests: The authors declare no conflict of interest.

Background: Genetic tests are increasingly performed for the management of unresectable pancreatic cancer. For genotyping aimed samples current guidelines recommend using core specimens, although based on moderate quality evidence. However, in clinical practice among the endoscopic ultrasound (EUS) guided tissue acquisition methods, fine needle aspiration (FNA) is the most widely performed., Aim: To assess the adequacy for next generation sequencing (NGS) of the DNA yielded from EUS-FNA pancreatic adenocarcinoma (PDAC) samples., Methods: Between November 2018 and December 2021, 105 patients with PDAC confirmed by EUS-FNA were included in the study at our tertiary gastroenterology center. Either 22 gauge (G) or 19G FNA needles were used. One pass was dedicated to DNA extraction. DNA concentration and purity (A260/280, A260/230) were assessed by spectrophotometry. We assessed the differences in DNA parameters according to needle size and tumor characteristics (size, location) and the adequacy of the extracted DNA for NGS (defined as A260/280 ≥ 1.7, and DNA yield: ≥ 10 ng for amplicon based NGS, ≥ 50 ng for whole exome sequencing [WES], ≥ 100 ng for whole genome sequencing [WGS]) by analysis of variance and t -test respectively. Moreover, we compared DNA purity parameters across the different DNA yield categories., Results: Our cohort included 49% male patients, aged 67.02 ± 8.38 years. The 22G needle was used in 71% of the cases. The DNA parameters across our samples varied as follows: DNA yield: 1289 ng (inter quartile range: 534.75-3101), A260/280 = 1.85 (1.79-1.86), A260/230 = 2.2 (1.72-2.36). DNA yield was > 10 ng in all samples and > 100 ng in 93% of them (one sample < 50 ng). There were no significant differences in the concentration and A260/280 between samples by needle size. Needle size was the only independent predictor of A260/230 which was higher in the 22G samples ( P = 0.038). NGS adequacy rate was 90% for 19G samples regardless of NGS type, and for 22G samples it reached 89% for WGS adequacy and 91% for WES and amplicon based NGS. Samples with DNA yield > 100 ng had significantly higher A260/280 (1.89 ± 0.32 vs 1.34 ± 0.42, P = 0.013). Tumor characteristics were not corelated with the DNA parameters., Conclusion: EUS-FNA PDAC samples yield DNA adequate for subsequent NGS. DNA amount was similar between 22G and 19G FNA needles. DNA purity parameters may vary indirectly with needle size., Competing Interests: Conflict-of-interest statement: All authors confirm they have no conflict of interest in relation to this manuscript., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

The selection of a DNA extraction method is a critical step when subsequent analysis depends on the DNA quality and quantity. Unlike mammals, for which several capable DNA extraction methods have been developed, for molluscs the availability of optimized genomic DNA extraction protocols is clearly insufficient. Several aspects such as animal physiology, the type (e.g., adductor muscle or gills) or quantity of tissue, can explain the lack of efficiency (quality and yield) in molluscs genomic DNA extraction procedure. In an attempt to overcome these aspects, this work describes an efficient method for molluscs genomic DNA extraction that was tested in several species from different orders: Veneridae, Ostreidae, Anomiidae, Cardiidae (Bivalvia) and Muricidae (Gastropoda), with different weight sample tissues. The isolated DNA was of high molecular weight with high yield and purity, even with reduced quantities of tissue. Moreover, the genomic DNA isolated, demonstrated to be suitable for several downstream molecular techniques, such as PCR sequencing among others.

DNA extraction from frozen blood clots is challenging. Here, the authors applied QIAGEN Clotspin Baskets and the Gentra Puregene Blood Kit for DNA extraction to cellular fraction of 5.5 ml whole blood without anticoagulating additives. The amount and quality of extracted DNA were assessed via spectrophotometer and gel electrophoresis. Results from array-based genotyping were analyzed. All steps were compared with DNA isolated from anticoagulated blood samples from a separate study. The quality and concentration of DNA extracted from clotted blood were comparable to those of DNA extracted from anticoagulated blood. DNA yield was on average 27 μg per ml clotted blood, with an average purity of 1.87 (A260/A280). Genotyping quality was similar for both DNA sources (call rate: 99.56% from clotted vs 99.49% from anticoagulated blood).

Forensic DNA analysis of cartridges and fired cartridge casings remains challenging, possibly due to the heat and pressure generated during firing of the weapon as well as metal ions from the casings that have been suggested to initiate DNA degradation and inhibit PCR during the DNA profiling process. Even though recently developed DNA recovery protocols have shown to significantly improve DNA yields and DNA profile success rates no information is available on whether the time interval between contact and the DNA recovery process has an influence on these outcomes. In the current study 40 cartridges and 40 fired cartridge casings were left untreated for 24 h or 1 week after which the rinse-and-swab technique was used to collect DNA. Higher DNA yields and higher DNA profile success rates were obtained from cartridges compared to fired cartridge casings. The same general observation was made when cartridges and fired cartridge casings were processed after 24 h compared to after 1 week. In addition, DNA profiles suitable for comparison could still be generated from samples when real-time PCR quantification indicated DNA concentrations < 0.001 ng/μl, suggesting that quantification results may not be reliable when assessing the presence of DNA on such items. In conclusion, the results indicate that cartridges and fired cartridge casings should be processed for DNA profiling as soon as possible and that DNA quantification results should be interpreted with caution as DNA profiles suitable for comparison could be missed. [ABSTRACT FROM AUTHOR]

BACKGROUND Cytology smears and cytospin preparations are increasingly being used for molecular testing. With these limited samples, optimizing tissue extraction to maximize the DNA yield is, therefore, critical. This study examined 2 common methods of tissue extraction and compared DNA yields from different types of glass slides. METHODS The H226 lung cancer cell line and 5 clinical samples of cellular effusions were used to prepare Diff-Quik-stained cytospins on 4 types of glass slides: fully frosted (FF), nonfrosted (NF), positively charged (PC), and silane-coated (SC). Tissue extraction was performed by either scalpel-blade scraping or cell lifting with the Pinpoint Slide DNA Isolation System (Zymo Research). DNA was extracted with the QIAamp DNA Mini Kit (Qiagen) and was quantified with the Quant-iT PicoGreen Kit (Life Technologies). RESULTS The DNA yield in cell-line cytospins was significantly lower from FF slides versus NF, PC, and SC slides with both scraping and cell-lifting methods. In addition, scraping yielded significantly more DNA than cell lifting ( P = .005). DNA yields from 5 clinical effusion cases with FF and NF slides showed results similar to the results for cell-line samples, with scraping consistently yielding more DNA than cell lifting and with NF slides outperforming FF slides. CONCLUSIONS Optimizing the DNA yield extracted from cytology specimens maximizes the chances of successful molecular testing and is critical in cases of low or marginal cellularity. This study demonstrates the following: 1) scraping yields more DNA than cell lifting, and 2) NF slides yield more DNA than FF slides. Cancer Cytopathol 2016;124:254-60. © 2015 American Cancer Society. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fires slightly affected the soil abiotic properties. • Holm oak favoured the microbial populations involved in N cycle in the unburnt area. • The abundances of eubacteria, nitrifiers and denitrifiers increased in the burnt area. • Fires hid the role of plant covers on soil microbial abundances. • A faster recovery of soil microbial populations was observed under pine and holm oak. The Mediterranean area is a fragile semi-arid ecosystem, characterized by a high level of biodiversity. Due to its climatic conditions, fires are frequent. Fires strongly impact the microbial community of the soil and the biogeochemical cycles of the ecosystem. Although the N cycle is crucial, limited data is available about the effects of fires on the microbial community involved in this cycle in these types of environments. The aim of this research was to evaluate the effects that fire has on the amount of microbial populations involved in the different steps of the N cycle in soils under different vegetation cover. To achieve this, surface soils were collected from unburnt and burnt soil of four plant species (holm oak, pine, black locust, and herbs) from inside the Vesuvius National Park that are typical of the Mediterranean maquis. The soils were analyzed for their main abiotic proprieties (pH, water content, concentrations of organic C, total C, total N, NH 4 +, NO2–, and NO3–). They were also analyzed for their amount of total DNA (DNA yield), eubacterial DNA (16S rDNA), N 2 -fixer, ammonia oxidizer, archaea ammonia oxidizer, and denitrifier DNA. The largest amount of microbial populations involved in the N cycle of the unburnt soil was observed in the holm oak soil. Fires slightly affected the soil abiotic properties and the DNA yield of the unburnt soil, but significantly increased the amount of eubacteria, nitrifiers, and denitrifiers. The conditions of the pine and holm oak soil encouraged a faster recovery of the amount of microbial populations involved in the N cycle. [ABSTRACT FROM AUTHOR]

BACKGROUND BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples. METHODS This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 10+ cells on a ThinPrep slide: 1+ (1-5 groups), 2+ (6-10 groups), 3+ (11-20 groups), and 4+ (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis. RESULTS Approximately 49% of thyroid FNA samples had low cellularity (1-2+). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 1+ to 4+ cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 10+ cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells. CONCLUSIONS This study demonstrates that by using 6 groups of 10+ follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens. Cancer (Cancer Cytopathol) 2014;122:114-22 © 2013 American Cancer Society. [ABSTRACT FROM AUTHOR]

Simple Summary: In the era of personalized oncology, next-generation sequencing plays an important role in identifying mutations that may predict the molecular pathomechanism and manage biliary tract cancers (BTC) therapy. The peripheral blood of cancer patients represents variable amounts of cell-free DNA (cfDNA) released from the tumor. Tumor-derived cfDNA in BTCs also allows the effective monitoring of the molecular genetic profile and the response to chemotherapy. Our study aimed to identify genetic aberrations in cell-free and matched tumor DNA in BTCs. We assume that the efficacy of the LB-based sequencing provides a novel perspective for BTCs therapy. Biliary tract cancer (BTC) is a rare malignancy with a long disease course and an overall poor prognosis. Despite multiple chemotherapy agents, there is no defined second-line treatment opportunity for advanced BTCs. In the era of precision oncology, NGS plays an important role in identifying mutations that may predict the molecular pathomechanism and manage the BTC therapy. The peripheral blood liquid biopsy (LB) of cancer patients represents variable amounts of cell-free DNA (cfDNA) released from tumor foci of any anatomical location. Our study aimed to identify somatic mutations and tumor variant burden (TVB) in cell-free and matched tumor DNA. We found a positive correlation between the estimated tumor volume and cfDNA yield (r = 0.9326, p < 0.0001). Comparing tissue and LB results, similar TVB was observed. SNVs were proven in 84% of the cases, while in two cases, only the LB sample was informative for molecular analysis. The most important aberrations in BTCs, such as FGFR2, IDH1, IDH2, KRAS, and TP53, could be detected in matched LB samples. Our prospective study demonstrates a minimally invasive testing approach to identify molecular genetic alterations in cholangiocarcinoma and gallbladder cancers. Clinical applications of cfDNA reflect by capturing the outstanding spatial tumor heterogeneity and guarantee novel aspects for the precision oncology treatment. [ABSTRACT FROM AUTHOR]

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

Abstract: We attempted to quantify microbial growth in soil by means of DNA determination after glucose amendment. An F DNA conversion factor of 5.0 was used to convert μg DNA g−1 soil to μg C mic g−1 soil during the growth phase. The conversion factor acquired rested on a regression analysis between soil microbial biomass-C (C mic) estimated by the substrate-induced respiration technique (SIR) and dsDNA using a modified, miniaturized dsDNA extraction procedure which included 44 field and forest soils with a coefficient of determination of r 2 = 0.95. Verification of this conversion factor was tested on eight arable soils where C mic was determined by substrate-induced respiration (SIR)-, chloroform fumigation-incubation (CFI)-, chloroform fumigation-extraction (CFE)-, and application of the F DNA conversion factor. The congruency between the C mic values obtained through these different techniques was satisfactory since five of eight soils gave similar C mic values which were not statistically significantly different. The soils were thereafter amended with glucose and microbial growth followed by C mic determinations with CFI, CFE, and DNA conversion over a period of up to 264 h at 22 °C. Concomitant CO2 analyses gave clues to two kinds of growth processes with respect to speed. Based on DNA conversion the calculated traditional growth parameters such as the specific growth rate (μ) lay in the range between 0.0046 and 0.022 h−1 which is several fold slower than μ values based on CO2 conversion but are in accordance with data in the earlier literature on growth rates for bacteria and fungi in soil done with traditional plate counts. These results suggest that DNA determinations can be applied as an alternative index for growth studies in situ. [Copyright &y& Elsevier]

Abstract: The study of microbial life in building materials is an emerging topic concerning biodeterioration of materials as well as health risks in houses and at working places. Biodegradation and potential health implications associated with microbial growth in our residues claim for more precise methods for quantification and identification. To date, cultivation experiments are commonly used to gain insight into the microbial diversity. Nowadays, molecular techniques for the identification of microorganisms provide efficient methods that can be applied in this field. The efficiency of DNA extraction is decisive in order to perform a reliable and reproducible quantification of the microorganisms by qPCR or to characterize the structure of the microbial community. In this study we tested thirteen DNA extraction methods and evaluated their efficiency for identifying (1) the quantity of DNA, (2) the quality and purity of DNA and (3) the ability of the DNA to be amplified in a PCR reaction using three universal primer sets for the ITS region of fungi as well as one primer pair targeting the 16S rRNA of bacteria with three typical building materials — common plaster, red brick and gypsum cardboard. DNA concentration measurements showed strong variations among the tested methods and materials. Measurement of the DNA yield showed up to three orders of magnitude variation from the same samples, whereas A260/A280 ratios often prognosticated biases in the PCR amplifications. Visualization of the crude DNA extracts and the comparison of DGGE fingerprints showed additional drawbacks of some methods. The FastDNA Spin kit for soil showed to be the best DNA extraction method and could provide positive results for all tests with the three building materials. Therefore, we suggest this method as a gold standard for quantification of indoor fungi and bacteria in building materials. [Copyright &y& Elsevier]

Abstract: The purpose of the study was to assess the suitability of three DNA extraction methods (Fast ID, SDS-based and relatively high-throughput methods) for single seeds (kernels) of canola, flax and broken pieces of soybean seeds. The extracted DNA was used for conventional and real-time qualitative PCR detection of the biotech events OXY235 canola, FP967 flax and DP305423 soybean. The mean weight of single canola and flaxseeds ranged from 2.3 to 3.2 mg and 5.5 to 5.8 mg, respectively. For canola, mean total DNA yields of 216, 796 and 377 ng per seed were obtained for Fast ID, SDS and relatively high-throughput DNA extraction methods, respectively. For flax, mean total DNA yields of 329, 826 and 957 ng per seed were obtained for Fast ID, SDS and relatively high-throughput DNA extraction methods, respectively. For soybean, mean DNA yields of 58, 102 and 175 ng per mg of broken seed were obtained for Fast ID, SDS and relatively high-throughput DNA extraction methods, respectively. Comparison of mean DNA yields indicated statistically significant differences among the three DNA extraction methods for all the three grain types. There was either weak or no correlation between seed weight and DNA yield for all the three DNA extraction methods. Abs260/280 ratio of ≥1.8 was obtained for DNA extracted with Fast ID and SDS-based methods. DNA isolated with all three extraction methods had low Abs260/230 ratios indicating the presence of contaminants that absorb at 230 nm. Consistent and repeatable PCR amplification was obtained for DNA extracted with the Fast ID and SDS-based extraction methods. Inhibition of PCR was observed for flax DNA extracted using relatively high-throughput method; however, reducing the amount of DNA to 10 ng in the PCR resulted in consistent amplification. Overall, all three DNA extraction methods can be used for fast DNA-based detection of unapproved or discontinued biotech events in single seeds of canola and flax as well as broken pieces of soybean seeds. [Copyright &y& Elsevier]