High-throughput DNA Isolation Research Articles

Development of a high-throughput DNA isolation method for livestock and poultry meat based on mesoporous metal-organic framework-coated solid-phase microextraction device

A rapid, portable, simple, low-cost, and high-throughput DNA isolation method was developed for the quantitative identification of meat adulteration based on mesoporous UiO-66 coated solid phase microextraction (SPME) devices. The high-throughput DNA extraction system consists of 96 UiO-66 SPME devices and a polyformaldehyde plate, which can extract DNA from 96 livestock and poultry meat samples simultaneously. At the same time, several parameters of the high throughput DNA isolation method were optimized, including lysis buffer volume, elution buffer volume, lysis time and elution time, etc. Compared with the single DNA isolation device, the 96-SPME high-throughput extraction system maintains the original good universality, reproducibility, chemical stability, and reusability. On this basis, it obtained higher DNA concentration (80 ± 34 ng μL−1), satisfactory DNA purity (A260/A280=2.4) and shorter extraction time (less than 7.2 s per sample), which are much more convenient and efficient than the conventional kit method and the single SPME DNA isolation method. In addition, the conventional multiplex polymerase chain reaction and real-time polymerase chain reaction techniques were used for the qualitative and quantitative detection of meat adulteration. The DNA obtained from this extraction method is highly reproducible and sensitive for the identification of duck ingredients in beef, with a detection range of 1–100 % and a minimum detection limit of 1 % (w/w).

Choice of gDNA isolation method has a significant impact on average murine Telomere Length estimates

Telomere Length (TL) and integrity is significantly associated with age-related disease, multiple genetic and environmental factors. We observe mouse genomic DNA (gDNA) isolation methods to have a significant impact on average TL estimates. The canonical qPCR method does not measure TL directly but via the ratio of telomere repeats to a single copy gene (SCG) generating a T/S ratio. We use a monochromatic-multiplex-qPCR (mmqPCR) method which multiplexes the PCR and enables quantification of the target and the single copy gene within the same qPCR reaction. We demonstrate that TL measurements, from murine gDNA, isolated via Spin Columns (SC) and Magnetic Beads (MB), generate significantly smaller T/S ratios compared to gDNA isolated via traditional phenol/chloroform methods. The former methods may impede correct TL estimation by producing non representative fragment sets and reducing qPCR efficacy. This work highlights discrepancies in TL measurements due to different extraction techniques. We recommend the use of gDNA isolation methods that are shown to preserve DNA length and integrity, such as phenol/chloroform isolation. We propose that widely used high throughput DNA isolation methodologies can create spurious associations within a sample set, thus creating misleading data. We suggest that published TL associations should be revisited in the light of these data.

High-Throughput DNA Isolation in Vegetable Crops for Genomics Applications.

Isolating high-quality DNA is essential for several applications in molecular biology and genomics. Performing whole-genome sequencing in crops and development of reduced representation genomic libraries for genotyping require precise standard on DNA in terms of concentration and purity. For screening large populations it is essential to increase the extraction throughput at affordable costs. In this chapter a homemade protocol is provided that is able to isolate in 96-well plates 198 samples of DNA in a single extraction. The method has been validated in tomato and pepper and can be applied in several vegetable species.

English

The extraction of high-quality DNA from okra (Abelmoschus esculentus L. Moench) is notoriously troublesome due to the high contents of polysaccharides, polyphenols, and different secondary metabolites. We have tested seven extraction buffers on silica dried okra leaves. Here, we describe a simple, rapid and modified procedure for high-quality DNA extraction from okra, which is amenable for downstream analyses. In contrast to Cetyl-trimethyl-ammonium bromide (CTAB) methods, the described procedure is rapid, omits the use of liquid nitrogen, phenol, PVP-10, and chloroform. It also uses inexpensive and less hazardous reagents and requires only ordinary laboratory equipment. The procedure employed a high concentration of Sodium dodecyl sulphate (SDS) to rid the problems associated with polysaccharides and polyphenols. The average yield was between 36 and 45 μg of total DNA from 90 mg of dried leaf weight. The DNA is adequate for molecular analysis of okra, such as genetic mapping or marker-assisted plant breeding. This protocol can be performed in as little as 3 h and may be adapted to high-throughput DNA isolation.   Key words: PVP-10, polyvinylpyrrolidone, non cetyltrimethylammonium bromide, okra, genomic DNA, Sodium dodecyl sulphate (SDS).

A modified protocol for rapid DNA isolation from cotton (Gossypium spp.)

Extraction of high-quality DNA from Gossypium (Cotton) species is notoriously difficult due to high contents of polysaccharides, quinones and polyphenols other secondary metabolites. Here, we describe a simple, rapid and modified procedure for high-quality DNA extraction from cotton, which is amenable for downstream analyses. In contrast to other CTAB methods, the described procedure is rapid, omits the use of liquid nitrogen, phenol, CsCl gradient ultracentrifugation, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use of PVP-10 to rid the problems associated with polysaccharides and polyphenols, respectively. The average yield was approximately 10–15 μg of good quality DNA from 100 mg of tissue weight, which is adequate for projects, like genetic mapping or marker-assisted plant breeding. This protocol can be performed in as little as 3 h and may be adapted to high-throughput DNA isolation.•Buffers A and B were redesigned from Paterson et al. (1993) and Porebski et al. (1997), respectively.•Ribonuclease A was added before chloroform extraction.•A simple, rapid and inexpensive DNA extraction method is described.

DNA extraction from hydatid cyst protoscolices: Comparison of five different methods.

Aim:The current study aimed to find out a simple, practical and high throughput DNA isolation method for extraction of DNA from hydatid cyst samples.Materials and Methods:Cattle and sheep isolate of hydatid cysts were obtained from the slaughterhouse, and hydatid fluid and protoscolices were collected in a sterile condition. Protoscolices were washed, 3 times with phosphate buffered saline, and DNA was extracted by different methods including manual extraction with freeze/thawing and phenol-chloroform, Triton X-100 extraction, and by a commercial kit (YTA, Yekta Tajhiz Azma, Iran) with three different modifications in the kit’s manufacturer instructions. The obtained DNA from the different methods was evaluated by Nanodrop in terms of the yield of DNA and carbohydrates or protein contaminations. To compare the quality of the extracted DNA, two pieces of the mitochondrial genome of Echinococcus granulosus, cox1, and nad1, were polymerase chain reaction (PCR)-amplified, using each of the DNA prepared by different methods. Electrophoresis of PCR products was carried out on the agarose gel.Results:The DNA extracted by manual method, using phenol/chloroform, had the highest yield, yet with the highest level of protein and carbohydrate contamination. The DNA extracted using two-step incubations, initially at 60°C for 2 h and then overnight at 37°C, was the most purified DNA with the lowest rate of contamination.Conclusion:Findings of the study demonstrated that modification in the currently available commercially DNA extraction kit resulted in the development of a high throughput DNA isolation method. This method can be recommended for the extraction of DNA from hydatid cysts, especially the cattle isolate where the extraction of DNA in these samples are usually problematic.

Modified Method of High Quality Genomic DNA Extraction from Mungbean [Vigna radiata(L.) Wilczek] Suitable for PCR Based Amplification

Objectives: Mungbean is one among the foremost necessary pulse crops. It is necessary to extract good quality genomic DNA for molecular work in plant science. Hence, present study intended to establish a robust and cost effectively genomic DNA extraction method for mungbean. Methods/Statistical Analysis: The prevailing CTAB method for genomic DNA extraction from plant was altered by avoiding usage of liquid nitrogen or lyophilization. Genomic DNA of plant was extracted from leaves of seven mungbean genotypes by grinding in pre-heated CTAB extraction buffer. Increased concentration of CTAB, PVP, β-mercaptoethanol, use of RNase treatment and repetition of purification step with C:I after P:C:I treatment will suppress the interference of the contaminants. The effectiveness of DNA extraction protocol for PCR amplification was evaluated using SSR and RAPD markers. Findings: The quality of DNA was assured by the presence of intact bands of genomic DNA separated on 0.8% agarose gel. The yield of genomic DNA ranged from 871.6 to 1143.2 ng/μL with an average of 979.14 ng/μL. When measured spectrophotometrically, the A 260 /A 280 ratio was in the range of 1.78 to 1.86, with an average of 1.82, indicating very low levels of contamination such as polysaccharides, phenolics and protein. The results of PCR amplification using SSR and RAPD markers indicated that the DNA extracted by this modified protocol was of decent quality and appropriate for various molecular studies. This protocol is suitable for underdeveloped countries, where resources for high throughput DNA isolation are not or less available. Application/Improvement: An effective, simple and cost-effective protocol for genomic DNA extraction with good yield as well as high purity was developed for mungbean which can be applied to other pulse crops also.

A Fast and Inexpensive High-Throughput Protocol for Isolating High Molecular Weight Genomic DNA from Lichens

Abstract: Werth, S., Reynisdottir, S., Guðmundsson, H. & Andresson, O. S. 2016. A fast and inexpensive high-throughput protocol for isolating high molecular weight genomic DNA from lichens. — Herzogia 29: 610–616. Isolating high molecular weight DNA as required for genomic library preparations and other applications is a challenging task. We optimized a glass-fiber column-based, high throughput DNA isolation protocol to yield comparatively large quantities of high molecular weight, pure DNA; cell lysis based on a CTAB buffer was followed by centrifugation to remove cell debris, binding to a glass-fiber membrane under high concentration of a chaotropic salt (guanidine hydrochloride), followed by two 70 % ethanol washes and elution. This method was utilized successfully on several species of lichenized ascomycetes and on two non-lichenized basidiomycetes. Thus, while developed for lichens, the method is suitable for a range of fungal taxa.

Stacking of five favorable alleles for amylase content, fragrance and disease resistance into elite lines in rice (Oryza sativa) by using four HRM-based markers and a linked gel-based marker

Marker-assisted selection (MAS) for qualitative traits such as grain quality and resistance to certain diseases has proven to be highly effective. Multiple genes responsible for various quality components and disease resistances can be simultaneously stacked to boost the performance and to lengthen the commercial lifespan of high-yield varieties. Grain quality genes (fgr and Wx) and three disease resistance genes (Pita, Pik and Xa23) have been well characterized and used in MAS breeding. However, stacking all of them together into a single variety has not been reported. We reported here the stacking of the five genes into elite lines in rice. We achieved this through the development of functional markers from causal mutations at fgr, Wx and Pita, a gene-targeted marker at Pik and the use of a linked marker for Xa23. We employed and optimized the high-resolution melting (HRM) analysis method for use as the genotyping platform of fgr, Wx, Pik and Pita. By combining high-throughput DNA isolation, multiplex and nested-PCR methods, we showed that HRM could serve as cost-effective, highly automated, moderate-throughput and reliable non-gel genotyping platform for a small-scale MAS program.

The effect of an enzymatic bone processing method on short tandem repeat profiling of challenged bone specimens

Forensic analysis of DNA from bone can be important in investigating a variety of cases involving violent crimes and mass fatality cases. To remove the potential presence of co-mingled remains and to eliminate contaminants that interfere with forensic DNA analysis, the outer surface of the bone fragment must be cleaned. This study evaluated two methods for processing bone specimens prior to DNA isolation. Mechanical sanding and enzymatic trypsin methods were compared in this study. The effects of these methods on the yield of DNA isolated and the quality of DNA analysis were studied. It was revealed that comparable values of DNA yields between the two methods were observed. Additionally, to evaluate the capabilities of the cleaning effect of the bone processing methods, the presence of polymerase chain reaction inhibitors in the DNA extracts was monitored using the internal positive control. Similar Ct values of the internal positive control were observed as the DNA extracts of the trypsin method compared with that of the sanding method. The characterization of the effects of the trypsin treatment on the quality of DNA profiling was also carried out. To evaluate the integrity of the nuclear DNA isolated, the percentage of allele calls and the peak-height values of alleles of the short tandem repeat profiles were compared between the two methods. A paired-sample t-test revealed no significant difference between the two methods. Our data suggested that the trypsin method can be used as an alternative cleaning method to mechanical cleaning methods. This method can be used to process multiple samples simultaneously. This can be very important for achieving high-throughput DNA isolation through potential automation, which can be extremely valuable for situations such as the forensic DNA analysis of skeletal remains from mass fatality incidents.

A high throughput DNA extraction method with high yield and quality

BackgroundPreparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome), and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L.) Moench] leaves and dry seeds with high yield, high quality, and affordable cost.ResultsWe developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles.ConclusionA high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

High-throughput DNA isolation method for detection of Xylella fastidiosa in plant and insect samples

We report an inexpensive, high-throughput method for isolating DNA from insect and plant samples for the purpose of detecting Xylella fastidiosa infection. Existing methods often copurify inhibitors of DNA polymerases, limiting their usefulness for PCR-based detection assays. When compared to commercially available kits, the method provides enhanced pathogen detection at a fraction of the cost.

Automated high throughput DNA isolation for detection of human papillomavirus in oral rinse samples

Background Oral HPV infection elevates risk of oropharyngeal cancer, but its natural history is unknown. Natural history studies necessitate validation of an automated, high-throughput method for HPV genomic DNA detection in oral rinse samples (ORS). Objectives To compare agreement of oral HPV detection in ORS processed by a magnetic-bead based automated platform to a previous gold-standard, manual protein-precipitation method. Agreement was compared to that of repeat sampling and repeat HPV testing. Study design HIV-infected individuals ( n = 100) provided two ORS collected 15 min apart. DNA was isolated from equal aliquots by either a protein-precipitation based (Puregene, Qiagen) or magnetic bead-based (QIAsymphony™ SP, Qiagen) method. HPV DNA was detected and type-specified by consensus primer PCR and reverse line blot hybridization. The kappa statistic was used to assess overall agreement (OA) and agreement on a positive test (Ps+). Results The DNA purification methods had very high agreement for categorizing an individual as HPV infected (OA = 0.95; Ps+ = 0.94) as well as for detection of HPV type-specific infection (OA = 0.99; Ps+ = 0.88) in ORS. Agreement for detection of HPV type-specific infection was greater than that observed with repeat oral rinse sampling (OA = 0.99, Ps+ = 0.76) but comparable to inter-assay agreement (OA = 1.00, Ps+ = 0.90). Conclusions HPV detection in ORS processed with a magnetic-bead based automated platform will facilitate large natural history studies of oral HPV infection necessary to evaluate the potential use of oral HPV detection in oral cancer screening.

DNA Extraction from Dried Plant Tissues Using 96-well Format (cTab Method)

This high throughput DNA isolation protocol is used to extract DNA of high quality from plant tissues for various genetics studies, like genotyping, and mapping etc. This protocol uses the well-established CTAB extraction procedure, and has been adapted to be used with 96-well plates., 这种高通量DNA分离方案用于从植物组织中提取高质量的DNA，用于各种遗传学研究，如基因分型和作图等。该方案使用已建立的CTAB提取方法，并已适用于96- 孔板。

Protocol: A high-throughput DNA extraction system suitable for conifers

BackgroundHigh throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA isolation from conifers.ResultsWe have developed a high-throughput DNA extraction protocol for conifers using an automated liquid handler and modifying the Qiagen MagAttract Plant Kit protocol. The modifications involve change to the buffer system and improving the protocol so that it almost doubles the number of samples processed per kit, which significantly reduces the overall costs. We describe two versions of the protocol: one for medium-throughput (MTP) and another for high-throughput (HTP) DNA isolation. The HTP version works from start to end in the industry-standard 96-well format, while the MTP version provides higher DNA yields per sample processed. We have successfully used the protocol for DNA extraction and genotyping of thousands of individuals of several spruce and a pine species.ConclusionA high-throughput system for DNA extraction from conifer needles and seeds has been developed and validated. The quality of the isolated DNA was comparable with that obtained from two commonly used methods: the silica-spin column and the classic CTAB protocol. Our protocol provides a fully automatable and cost effective solution for processing large numbers of conifer samples.

A simple and affordable method for high‐throughput DNA extraction from animal tissues for polymerase chain reaction

We have developed a very simple and inexpensive method for high-throughput DNA extraction from animal tissues. The procedure contains three steps (digestion, heating, and centrifugation) and it is compatible with the 96-well plate format commonly used in polymerase chain reaction (PCR) amplifications. The duration for processing a plate is about 1.5 h; therefore, one researcher can isolate DNA from up to 1000 samples during a single workday. A small piece of tissue (ca. 10-20 mg) yields enough template for at least 50-70 PCR amplifications, as demonstrated by using the processed samples as templates successfully for long distance PCR, multiplex PCR, and randomly amplified polymorphic DNA (RAPD) assay. The application of our method is expected to facilitate studies that require high-throughput DNA isolation for PCR amplification, such as genotyping by microsatellites for mapping and genetic diversity studies, as well as mutant screening in zebrafish.

Pollen-mediated transgene flow in the wind-pollinated grass species tall fescue (Festuca arundinacea Schreb.)

Information regarding gene flow in wind-pollinated, outcrossing forage grasses is essential for any future releases of value-added transgenic cultivars. Experiments on pollen dispersal was carried out by growing transgenic tall fescue (Festuca arundinacea) in a central plot, surrounded by exclosures containing recipient plants up to a distance of 200 m from the central source plants in eight directions. The central transgenic tall fescue plants carried a chimeric hygromycin phosphotransferase gene (hph) and a chimeric β-glucuronidase gene (gusA). Seeds were collected from the recipient plants and germinated seedlings were used for high throughput DNA isolation and polymerase chain reaction (PCR) analysis. More than 21,000 seedlings were PCR analyzed for the experiments conducted in three years. Transgenes were detected in recipient plants at up to 150 m from the central transgenic plot. The highest transgene frequencies, 5% at 50 m, 4.12% at 100 m and 0.96% at 150 m, were observed north of the central plot, the prevailing wind direction. Lower transgene frequencies were detected in other directions, particularly at 100 m and 150 m distances. No transgene was detected at 200 m distance in any direction. Transgene flow was less effective or ineffective when recipient plants were further away from the central donor plants. Southern blot hybridization analysis confirmed the transgenic nature of the PCR positive plants. A supplementary experiment demonstrated that transgene flow can be controlled by placing transgenic plantings downwind and long distances from non-transgenic seed increases, thus allowing tall fescue breeding and transgene development programs to be conducted concurrently at the same research station.