Method For Extraction Of Genomic DNA Research Articles

Comparison study of three methods for genomic DNA extraction from fresh and herbarium leaf specimens of Achillea wilhelmsii C.Koch

Comparison study of three methods for genomic DNA extraction from fresh and herbarium leaf specimens of Achillea wilhelmsii C. Koch

A Modified Method for Rapid Genomic DNA Extraction from Tuberose

The isolation of high yield and quality genomic DNA is crucial for studying the molecular genetics of plants. However, high contents of secondary metabolites, especially in medicinal and aromatic plants, interfere with the extraction of clean DNA, thereby rendering it useless for downstream analyses such as DNA amplification, restriction, sequencing and cloning. The chemotypic heterogeneity among plant species may not permit high quality DNA isolation with a single protocol, thus a species-specific extraction method is required for quality extractions. Here, we present a modified cetyltrimethylammonium bromide (CTAB) protocol for good quality DNA extraction from tuberose, which is an important plant for perfume and pharmaceutical industry due to its pleasant fragrance and essential oil content. In contrast to other CTAB methods, the modified procedure is rapid, omits the use of liquid nitrogen and phenol, uses inexpensive and less hazardous reagents, and requires only ordinary laboratory equipment. The procedure employed the high concentration of NaCl and use ofPVP-10 to get rid of problems associated with polysaccharides and polyphenols, respectively. The yield and quality of extracted DNA were fairly good and amenable for downstream analyses. Moreover, to our knowledge, the described method is the first report of a modified DNA extraction protocol for tuberose.

An easily-performed high-throughput method for plant genomic DNA extraction

Genomic DNA isolation is a crucial technique for researchers studying plant molecular biology. A current widely-used protocol for DNA extraction needs a pestle and mortal for each sample and consumes a large amount of liquid nitrogen in grinding the samples. Most high-throughput methods depend on expensive machines for tissue homogenization. Here we developed a CTAB-based DNA extraction method using 2.0 mL microcentrifuge tubes for sample processing. This protocol has the advantages that it is suitable for a variety of plants, easily-performed without special equipment, and high-throughput; it effectively avoids sample cross-contamination, and is inexpensive, rapid and safe.

Determination of mir-155 and mir-146a expression rates and its association with expression level of TNF-α and CTLA4 genes in patients with Behcet’s disease

IntroductionMicroRNAs (miRNAs) are involved in the pathogenesis of inflammatory diseases. MiR-146 and miR-155 emerged as key regulators of the immune response. This study designed to analyze the miR-146a and miR-155 expression in patients with Behcet’s disease (BD) and investigated their association with the expression of tumor necrosis factor-alpha (TNF-α) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4) genes. MethodologyIn a case-control study, 47 Iranian Azeri BD patients and 61 age- and sex matched healthy controls recruited to the study. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA blood tubes by Ficoll density-gradient centrifugation. Genomic DNA samples of BD and healthy controls were extracted using the rapid genomic DNA extraction method from the peripheral blood collected in tubes containing EDTA. Total RNA was extracted from the PBMCs according to the TRIzol protocol. MiR-146a, miR-155, TNF-α and CTLA-4 expression were studied using real-time PCR. ResultsMiR-155 and TNF-α expression was significantly increased, whilst CTLA-4 expression was significantly decreased in the PBMCs of BD patients. There was no significant difference in the miR-146a expression rate between BD patients and controls. A positive correlation between miR-155 and TNF-α expression and negative correlation between miR-155 and CTLA-4 expression were observed. No significant association was observed between the expression of miR-155, miR-146a, TNF-α and CTLA-4 genes with BD activity. MiR-155 and miR-146a expression rate were significantly higher in patients with uveitis and phlebitis, respectively. Discussion and conclusionThe expression of miR-155 increased in BD and associated with upregulation of TNF-α and downregulation of CTLA-4 genes.

Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing

BackgroundThe recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or “orphan” crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6–$9/sample) for consumables.ResultsWe modified a non-organic solvent protocol to extract microgram quantities (1–13 μg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384–576 samples/day), and is inexpensive at US$0.62/sample for consumables.ConclusionsThis versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

A simple and rapid method for isolation of DNA for molecular markers and transgene analysis in safflower

In spite of the possibility of analysing large samples and the rapidity of Polymerase Chain Reaction (PCR) technology, the usual protocols for DNA plant extraction remain time-consuming, slow and involves use of hazardous chemicals. To lessen labor, time or cost of DNA extraction, a simple and instant method for genomic DNA extraction from leaf tissue of safflower (Carthamus tinctorius L.) leaf is established. Small quantity of tissue material (typically3-5mg) was ground in a centrifuge tube using plastic pestle in extraction solution. ExtractedDNA was suitable for PCR analysis, without centrifugation. The feasibility of this method was confirmed by testing molecular markers and transgene detection. This method requires less than 1 mg of plant tissue stored frozen or used fresh and is useful for molecular marker analyses as well as transgene detection.

Evaluation of Genomic DNA-Extraction Methods for Molecular Analysis of Solanecio biafrae

ABSTRACT 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.

A modified method for genomic DNA extraction from the fish intestinal microflora

A modified genomic DNA extraction method named the combination of lysozyme and ultrasonic lysis (CLU) method was used to analyze the fish intestinal microflora. In this method, the physical disruption and chemical lysis steps were combined, and some parameters in the key steps were adjusted. In addition, the results obtained by this method were compared with the results obtained by the Zirmil-beating cell disruption method and the QIAamp Fast DNA Stool Mini Kit. The OD260/OD280 ratio and concentration of the DNA extracted using the CLU method were 2.02 and 282.8 µg/µL, respectively; when the incubation temperatures for lysozyme and RNase were adjusted to 37 °C, those values were 2.08 and 309.8 µg/µL, respectively. On the agarose gel, a major high-intensity, discrete band of more than 10 kb was found for the CLU method. However, the smearing intensity of degraded DNA was lower when the incubation temperatures were 60 °C for lysozyme and 30 °C for RNase than when incubation temperatures of 37 °C for lysozyme and 37 °C for RNase were used. The V3 variable region of the prokaryotic 16S rDNA was amplified, and an approximately 600-bp fragment was observed when the DNA extracted using the CLU method was used as a template. The CLU method is simple and cost effective, and it yields high-quality, unsheared, high-molecular-weight DNA, which is comparable to that obtained with a commercially available kit. The extracted DNA has potential for applications in critical molecular biology techniques.

CTAB influenced differential elution of metagenomic DNA from saltpan and marine sediments.

A simple, reliable method for genomic DNA extraction from sediments with minimum contaminants was developed to address the risk of poor quality DNA in metagenomic studies. Nine DNA extraction methods using 20% cetyl-trimethyl-ammonium bromide (CTAB) were performed and compared to develop an extraction protocol that can offer humic acid-free metagenomic DNA from marine and saltpan sediments. Community DNA extraction was executed via., Zhou et al. modified protocol using 20% CTAB treatment at different steps to compare the efficacy of humic acid removal. Out of nine DNA extraction methods, method 6 significantly improved the quality of DNA with efficient removal of humic substances. 16S rRNA gene amplification and spectrophotometric analysis confirmed the efficiency of method 6 to remove DNA inhibitors from marine sediments as well as saltpan samples. Inhibitors extracted along with metagenomic DNA outcome increased DNA yield and PCR inhibition in method 1 and 3. However, repeated 20% CTAB wash in method 6 ensured 16S amplification and least yield and concentration. Current study explains a detailed protocol based on 20% CTAB wash for the extraction of humic acid-free DNA from diverse sediment samples.

A Modified CTAB Method for Genomic DNA Extraction from Apple Fruit

According to the high content of polysaccharides and polyphenols in apple fruits, three modified CTAB methods were used to extract genomic DNA of ‘Granny Smith’, with young leaves as control. The results showed that the method three was the best. This method integrated multiple purification steps, adding the interferent scavenger before cell lysis, increasing the concentration of β-mercaptoethanol and PVP to inhibite the oxidation of polyphenol, and adding 3 mol/L NaAC of 1/10 volume, which effectively removed the polysaccharides, polyphenols and other secondary metabolites. The results of ultraviolet spectrophotometer and agarose gel electrophoresis showed that DNA was high purity and good integrity. The results of ISSR primers amplification showed that the bands were clear. The results of the digestion of EcoR I restriction enzyme indicated that DNA could be digested by restriction enzyme. Therefore, the method three is the best for genomic DNA extraction. This method provides a reference for the removal of polysaccharides and polyphenols in the process of DNA extraction.

An optimised pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridisation.

ABSTRACTAdvances in genome engineering have resulted in the generation of numerous zebrafish mutant lines. A commonly used method to assess gene expression in the mutants is in situ hybridisation (ISH). Because the embryos can be distinguished by genotype after ISH, comparing gene expression between wild-type and mutant siblings can be done blinded and in parallel. Such experimental design reduces the technical variation between samples and minimises the risk of bias. This approach, however, requires an efficient method of genomic DNA extraction from post-ISH fixed zebrafish samples to ascribe phenotype to genotype. Here we describe a method to obtain PCR-quality DNA from 95-100% of zebrafish embryos, suitable for genotyping after ISH. In addition, we provide an image analysis protocol for quantifying gene expression of ISH-probed embryos, adaptable for the analysis of different expression patterns. Finally, we show that intensity-based image analysis enables accurate representation of the variability of gene expression detected by ISH and that it can complement quantitative methods like qRT-PCR. By combining genotyping after ISH and computer-based image analysis, we have established a high-confidence, unbiased methodology to assign gene expression levels to specific genotypes, and applied it to the analysis of molecular phenotypes of newly generated lmo4a mutants.

An Optimized CTAB Method for Genomic DNA Extraction from Freshly-picked Pinnae of Fern, Adiantum capillus-veneris L.

As the sister clade of seed plants, ferns are significant materials for plant phylogeny research. However, the genomic DNA extraction protocol for fern samples like modified CTAB method still lacks robustness. Here, we found that the amount and condition of the pinnae samples are critical for gDNA extraction in fern, Adiantum capillus-veneris L. In 500 μl CTAB solution, the recommended amount of pinnae is about 10-20 mg (2-3 pieces). The condition of the pinnae must be instantly-picked from a plant cultivated in a suitable environment. With these factors under control, it is highly reproducible to get the high-quality gDNA with low degradation rate.

Comparison of six genomic DNA extraction methods for molecular downstream applications of apple tree (Malus X domestica)

Extraction of high quality DNA is crucial for any molecular genetic analysis. However, it is difficult to be obtained from problematic plant tissue, high in phenolic compounds, such as apple leaves...

Fungal Genomic DNA Extraction Methods for Rapid Genotyping and Genome Sequencing.

Isolation of fungal genomic DNA of high quality is required for a number of downstream biotechnology-derived applications such as genome sequencing, microarrays, and digital PCR technologies, to only name a few. In most cases, not only a high molecular weight DNA of superior grade is required but also large quantities. On the other hand, a number of laboratory experiments, such as polymerase chain reaction (PCR) for medical diagnostic or for genotyping, have to be conducted in a limited amount of time and can provide complete results with the use of lower quality DNA. We describe here two different fungal DNA extraction approaches, which are applicable to a wide range of fungal species.First, we adapted a DNA extraction method for PCR-based genotyping which allows analysis of single to hundreds of colonies simultaneously. Cells are disrupted in the presence of sodium dodecyl sulfate and Proteinase K which are then removed by precipitation and centrifugation. The cleared lysate is used for PCR reaction.Secondly, we describe a method to obtain genome sequencing quality grade DNA from fungal liquid cultures. Mycelia are harvested by either filtration or centrifugation. Cells are mechanically disrupted by liquid nitrogen grinding, followed by genomic DNA extraction using the QIAGEN's DNeasy® Plant Kit. The quality and quantity of genomic DNA is monitored by fluorometry.

A simple, efficient and universal method for the extraction of genomic DNA from bacteria, yeasts, molds and microalgae suitable for PCR-based applications

The extraction of genomic DNA from microbial cells plays a significant role in PCR-based applications such as molecular diagnosis, microbial taxonomy, screening of genetically engineered microorganisms, and other such PCR-based applications. Currently, many methods for extraction of genomic DNA from microorganisms have been developed. However, these methods either require hazardous chemicals or consist of time-consuming steps for effective execution. In this study, we have established a simple and universal genomic DNA extraction method for different microorganisms including bacteria, yeasts, molds, and microalgae. Our method does not require harmful reagents such as phenol and chloroform for the extraction process to minimize the generation of hazardous wastes. The obtained genomic DNA products displayed high concentrations and represented a good purity level with the average 260 nm/280 nm absorbance ratios (A260/280) that range from 1.6 to 2.0. The DNA molecules further remained considerably intact when analyzed on agarose gels. More importantly, these DNA products were qualified through successful PCR amplifications of 16S rRNA gene, rDNA internal transcribed spacer (ITS), or 18S rRNA gene from genomes of bacteria, fungi, and microalgae respectively. Furthermore, with the extracted genomic DNA products, the processes of the identification of the haploid and diploid states of the Saccharomyces yeast strains or detection of putative strains of Aspergillus oryzae and Aspergillus flavus that have been isolated from infected food materials through PCR analyses are facilitated. The genomic DNA extraction method established in this study is easy to manage, time saving and cost-effective, and environmentally friendly.

Investigation of the Connexin-30 gene Deletion Mutations Del (GJB6-D13s1830) and Del (GJB6-D13s1854) among Subjects with Non-Syndromic Sensorineural Hearing Loss in Khorasan Razavi Province

Introduction: Hearing loss (HL) is the most common sensorineural disorder with the frequency of 1 to 1000 neonates and it's seen in two syndromic and non-syndromic states. Given that the mutations of DFNB1 locus, containing Connexin 26 (Cx26) and 30 (Cx30) genes are the only responsible for 50% non-syndromic HL with autosomal recessive hereditary in different populations, the aim of this study is the screening of the connexin30 gene deletion mutations including Del (GJB6-D13S1830) and Del (GJB6-D13S1854) in HL subjects were negative to mutation in Cx26 gene. Materials & methods: In this descriptive study, 85 unrelated patients (44 males and 41 females) with autosomal recessive hearing loss of Khorasan Razavi province that were negative towards the Cx26 gene mutations, were studied. Genomic DNA was extracted from 1 ml of EDTA anticoagulated peripheral blood by rapid genomic DNA extraction (RGDE) method. For molecular analysis, multiplex PCR and direct sequencing methods were used. Findings: Out of 170 studied chromosomes, none of the Del (GJB6-D13S1830) and Del (GJB6-D13S1854) mutations found in the studied subjects. Discussion & conclusions: Lack of the deletion mutations in the Cx30 gene, indicates the involvement of other genes in causing disease. Thus, it is necessary to study other HL genes in these subjects.

Rapid and Simple Method for the Extraction of Genomic DNA from Tobacco (Nicotiana tabacum L.) Seedlings

Abstract Tobacco (Nicotiana tabacum L.) plants are known to present high levels of secondary metabolites that increase with the plant age. Molecular biology techniques like restriction enzyme digestion and PCR, requires as pre-requisite the isolation of genomic DNA of suitable purity, good quality and with low levels of contaminants. Several methods to isolate pure and intact tobacco DNA for molecular research purposes have been developed. In this work, a combination between a tobacco seed germination technique using gibberellic acid and a fast and simple genomic DNA extraction method from 14-days old tobacco seedlings to reduce the secondary metabolites levels in the final samples was presented. Ten tobacco genotypes were used to evaluate this method. The DNA concentrations were in a range between 0.73 μg/μL to 1.47 μg/μL for Habana-2000 cv. and Criollo cv., respectively. The absorbance ratios values to determine DNA quality were acceptable. This method allows the obtaining of high molecular weight DNA suitable for digestion with restriction enzymes, EcoRI and BamHI. Tobacco seedlings DNA in a short period of time, in a simple way and with a low cost, was obtained with this extraction method.

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

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

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.

Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize

BackgroundThe world’s top three cereals, based on their monetary value, are rice, wheat, and corn. In cereal crops, DNA extraction is difficult owing to rigid non-cellulose components in the cell wall of leaves and high starch and protein content in grains. The advanced techniques in molecular biology require pure and quick extraction of DNA. The majority of existing DNA extraction methods rely on long incubation and multiple precipitations or commercially available kits to produce contaminant-free high molecular weight DNA.ResultsIn this study, we compared three different methods used for the isolation of high-quality genomic DNA from the grains of cereal crop, Zea mays, with minor modifications. The DNA from the grains of two maize hybrids, M10 and M321, was extracted using extraction methods DNeasy Qiagen Plant Mini Kit, CTAB-method (with/without 1% PVP) and modified Mericon extraction. Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain codes for 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS regions show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this study, the genomic DNA was then amplified with PCR using primers specific for ITS gene. PCR products were then visualized on agarose gel.ConclusionThe modified Mericon extraction method was found to be the most efficient DNA extraction method, capable to provide high DNA yields with better quality, affordable cost and less time.