Method For Extraction Of Genomic DNA Research Articles

A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using High Resolution Melting analysis.

BackgroundThe CRISPR/Cas9 system has become a regularly used tool for editing the genome of many model organisms at specific sites. However, two limiting steps arise in the process of validating guide RNA target sites in larvae and adults: the time required to identify indels and the cost associated with identifying potential mutant animals.ResultsHere we have combined and optimized the HotSHOT genomic DNA extraction technique with a two-steps Evagreen PCR, followed by a high-resolution melting (HRM) assay, which facilitates rapid identification of CRISPR-induced indels. With this technique, we were able to genotype adult zebrafish using genomic DNA extracted from fin-clips in less than 2 h. We were also able to obtain a reliable and early read-out of the effectiveness of guide RNAs only 4 h after the embryos were injected with the constructs for the CRISPR/Cas9 mutagenic system. Furthermore, through mutagenesis kinetic assay, we identified that the 2-cell stage is the earliest time point at which indels can be observed.ConclusionsBy combining an inexpensive and rapid genomic DNA extraction method with an HRM-based assay, our approach allows for high-throughput genotyping of adult zebrafish and embryos, and is more sensitive than standard PCR approaches, permitting early identification of CRISPR-induced indels and with applications for other model organisms as well.

GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA.

Existing yeast genomic DNA extraction methods are not ideally suited to extensive screening of colonies by PCR, due to being too lengthy, too laborious or yielding poor quality DNA and inconsistent results. We developed the GC prep method as a solution to this problem. Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin. In around 12 minutes, multiple samples can be processed to extract high yields of genomic DNA. These preparations perform as effectively in PCR screening as DNA purified by organic solvent methods, are stable for up to 1 year at room temperature and can be used as the template for PCR amplification of fragments of at least 8 kb.

Establishment of A Rapid and Inexpensive Identification Method for HLA-B*58: 01 Genotype

Abstract A new method for polymorphism detection of rs9263726 site on Psoriasis Susceptibility1 Candidate1 (PSORS1C1) was established based on the pyrosequencing technology and the improved method of genomic DNA extraction from whole blood. The detection limit of the method was 0.4 ng μL −1 genomic DNA. A total of 20 samples were detected by pyrosequencing, and the results were consistent with those of Sanger sequencing. By gene-sequencing of 683 clinical samples with pyrosequencing method, the polymorphism distribution of rs9263726 in Chinese was summarized as GG (87.6%), GA (11.7%), and AA (0.7%). Besides, 46 clinical samples were detected to analyze the relationship between rs9263726 genotype and HLA-B*5801 genotype, and the results showed that the sensitivity of the method was 100%, and the specificity was 91.3%. This study demonstrated a new method for HLA-B*58:01genotyping.

Single Lysis-Salting Out Method of Genomic DNA Extraction From Dried Blood Spots.

Dried blood spots (DBS) are an important form of bio-sampling and valuable approach for storing blood samples for genetic studies. This has necessitated in developing an effective protocol to isolate genomic DNA (gDNA) from DBS samples.In this study, we have elucidated a dependable and non-hazardous "single lysis-salting out" (SLSO) protocol of gDNA extraction from DBS and compared against the available commercial kits. For the purpose of this study, blood spots were collected on S&S 903 filter cards from 10 healthy volunteers and 30 patients with glutaric aciduria type I (GA-I). The gDNA was extracted from theseDBS samples by SLSO, QIAamp® gDNA Micro kit and innuPREP forensic kit methods. The quantity and quality of gDNA obtained from these methods were determined by measuring the absorbance using a Nanodrop spectrophotometer. The SLSO method showed four-fold and eight-fold increased yield of gDNA in healthy volunteers and patient samples, respectively, compared to commercial kits (p<0.0001). The protocol was also found to be cost efficient, reducing the per sample cost to almost half. The suitability of this method for genetic studies was confirmed by performing R402W genotyping by RFLP in GA-I patients. The genotyping results showed the presence of R402W mutation in 20% (6/30) of patients. The SLSO method was found to be inexpensive, non-hazardous and a suitable technique for isolating gDNA from DBS samples for genetic studies.

A rapid genomic DNA extraction method and its combination with helicase dependent amplification for the detection of genetically modified maize

A simple, solvent-free extraction method for DNA facilitates the analysis for genetically modified organisms.

Comparative Sensitivity and Specificity of Polymerase Chain Reaction Assays for the Detection of Theileria equi Coupled With Three DNA Template Extraction Methods

The present study demonstrates the relative sensitivity and specificity of two 18S rRNA gene-based polymerase chain reaction (PCR) assays (designated as PCR1 and PCR2) coupled with three genomic DNA extraction methods viz., via Whatman filter paper#1 method (Ext 1), Whatman Flinders Technology Associate (FTA) elute microcards method (Ext 2), and Qiagen DNeasy Blood & Tissue Kits (Ext three3) for the detection of Theileria equi. Both PCR assays on the nucleic acid of 72 field blood samples extracted by Ext 1 and Ext 3 revealed an prevalence rate of 2.77 and 18.56%, respectively; whereas in case of Ext 2, PCR1 and PCR2 revealed 16.67% and 18.56% prevalence, respectively. Ext 1 showed only 15.38% sensitivity and 100% specificity, whereas Ext 2 displayed 92.3% to 100% sensitivity and 100% specific results with respect to Ext 3. The amplified products were clearly positive with a diminishing signal up to 10−6 DNA dilutions in Ext 2 and Ext 3 method in both PCRs. Ext 1 was able to detect only 10−3 and 10−4 DNA dilution. Based on these results, it was concluded that blood sampled on FTA cards for DNA extraction is the recommended approach with detection ability up to 10−6 dilution.

Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue

As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and “amplifiability” for aCGH and other downstream applications. Phenol–chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues.

A comparison of DNA extraction methods for biodiversity studies of eukaryotes in marine sediments

There is increasing interest in understanding the diversity of eukaryotic microorgan- isms living in marine sediments, particularly to assess the effects of anthropogenic activities. Sequencing technologies generate high-resolution data for biodiversity studies that are useful for environmental monitoring. However, there are challenges in coupling classical monitoring with new sequencing technology and, consequently, there is a requirement for stringent optimization and standardization of any new protocol. Sample preparation is a critical factor because errors introduced during this step will severely affect further analyses and conclusions. This is particu- larly important in studies where biodiversity between different samples is compared, such as in environmental monitoring programs. Several protocols for extracting genomic DNA from soil and sediment samples have been developed, but most are optimized for prokaryotes in terrestrial soils and may therefore not be optimally adapted for investigating benthic marine eukaryotes. In this study, we compared existing and modified genomic DNA extraction methods on 2 different mar- ine sediments with the aim to find an optimal protocol for processing a high number of sediment samples for further sequencing analysis. The protocols were evaluated based on quantity and quality of genomic DNA and recovered biodiversity. Results indicated significant variations in overall genomic DNA yield and purity among protocols. Further, our data suggest an effect of genomic DNA extraction procedure on eukaryotic diversity profiles, particularly for sediments with a high content of silt and clay.

A method for fast genomic DNA extraction from saliva and its application

Objective To explore a fast,effective and noninvasive method of genomic DNA extraction.Methods We compared the genomic DNAs extracted by two DNA extraction methods of potassium iodide(KI) and Kit from saliva(fresh and placed a week at room temperature),and PCR amplifications were done for TP53 and PRB-3 genes from their extracted DNA.Ninety-nine healthy children and adults were recruited and their saliva samples were collected to detect the stability of DNA extracted by KI.Results High quality genomic DNAs were extracted by both methods from the fresh saliva.The DNA yield based on KI was(1.91 ± 0.15) μg,with the D260/ D280 being 1.99 ± 0.05,and that of Kit was(2.64 ± 0.34) μg,with the D260/ D280 being 1.81 ± 0.02.Although the extracted DNAs had obvious degradation from saliva samples placed for a week at room temperature,the expectant DNA fragments were successfully amplified by PCR for TP53 and PRB-3 genes using these extracted DNAs.Although the genomic DNAs from saliva samples of 99 healthy volunteers by the method of KI showed individual difference(DNA yield was [1.89 ± 0.46] μg),the DNA quality was stable and reliable(D260/ D280 was 1.96 ± 0.10).Conclusion KI method for extracting genomic DNA from saliva is not only cheap and highly effective,but also noninvasive,making it suitable for large-scale epidemiological studies.

A rapid and efficient method of fungal genomic DNA extraction, suitable for PCR based molecular methods

A rapid and efficient method of fungal genomic DNA extraction, suitable for PCR based molecular methods

Improved DNA Extraction Method for Molecular Diagnosis from Smaller numbers of Cells

Isolating total DNA from small samples using traditional methods is difficult and inefficient mainly due to loss of DNA during filtration and precipitation. With advances in molecular pathology, DNA extraction from micro-dissected cells has become essential in handling clinical samples. Genomic DNA extraction using small numbers of cells can be very important to successfully PCR amplify DNA from small biopsy specimens. We compared our experimental genomic DNA extraction method (A) with two other commercially available methods: using spin columns (B), and conventional resins (C), and determined the efficacy of DNA extraction from small numbers of cells smeared on a glass slide. Approximately 50, 100, 200, 500 and 1000 cells were isolated from fine needle aspiration biopsy (FNAB) slides aspirated from histologically proven papillary thyroid carcinoma masses. DNA was extracted using the three techniques. After measuring DNA quantity, PCR amplification was performed to detect the b-globin and BRAF V600E gene mutations. DNA extracted by method (A) showed better yield than the other methods in all cell groups. With our method, a suitable amount of genomic DNA to produce amplification was extracted from as few as 50 cells, while more than 100 to 200 cells were required when methods (B) or (C) were applied. Our genomic DNA extraction method provides high quality and improved yields for molecular analysis. It will be especially useful for paucicellular clinical samples which molecular pathologists often confront when handling fine needle aspiration cytology, exfoliative cytology and small biopsy specimens.

Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real-time PCR.

Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte-colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra-assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild-type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin-based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue-specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved.

Comparison of methods for high quantity and quality genomic DNA extraction from raw cow milk.

Isolation of sufficient quantities of high quality DNA is a prerequisite for molecular studies. Milk somatic cells can be used; however, inhibitors such as fats and proteins make milk a difficult medium for extracting large amounts of quality DNA. We optimized, evaluated and compared three methods, Modified Nucleospin Blood Kit method, Modified TianGen Kit method and Phenol-Chloroform method for genomic DNA extraction from bovine milk. Individual cows' milk and bulk milk samples were collected from a China agricultural university dairy farm. Genomic DNA extracted from each milk sample by the three methods was evaluated for quantity and purity by spectrophotometry and gel electrophoresis, as well as PCR and sequencing. All the three methods were found suitable for genomic DNA isolation from bovine milk, PCR applications, and sequencing. Comparing the three methods, we found that the Modified Nucleospin Blood Kit method was significantly better than the Phenol-Chloroform method in terms of quantity as well as quality (amount, concentration, 260/280 nm and 260/230 nm absorbance ratio), whereas, the Modified TianGen Kit method was more efficient than the Phenol-Chloroform method and cheaper than the Modified Nucleospine Blood Kit method; it yielded reasonably good quantities of good quality DNA and would be suitable for large-scale genotyping of lactating cows.

Molecular Identification of Mediterranean Mutation in Patients with Deficiency of Glucose-6-Phosphate Dehydrogenase (G6pd) in North West of Iran

Glucose-6-phosphate dehydrogenase (G6PD) is a highly polymorphic enzyme that is encoded by human Xlinked gene.G6PD deficiency is the most common enzymopathy in human with approximately 400 million people affected worldwide. According to the previous studies, Mediterranean mutation (C563T) is one of the most common G6PD enzyme mutations in Iranian population with a prevalence range of %63 to %86/4.This study was conducted in order to determine Mediterranean mutation, through molecular method ,in the population of the North-west of Iran. In this study, by Rapid Genomic DNA Extraction (RGDE) method, from 77 blood samples of unrelated male and female patients with genetic deficiency of G6PD, DNA was extracted and after digestion by MboII enzymes, in order to search for Mediterranean mutation, they were analyzed by means of PCR-RFLP and sequencing methods. Of the 77 patients, 48 patients (37 male, 11 female) had a Mediterranean mutation frequency of 62/3 %. 29 patients (37/7%) had mutations other than Mediterranean type. This study revealed that G6PD is the most common mutation in Iranian North-west population. Further studies are recommended to identify the mutation type of other varieties.

An improved method to extract DNA from mango Mangifera indica

High quality genomic DNA is the first step in the development of DNA-based markers for fingerprinting and genetic diversity of crops, including mango (Mangifera indica L.), a woody perennial. Poor quality genomic DNA hinders the successful application of analytical DNA-based tools. Standard protocols for DNA extraction are not suitable for mango since the extracted genomic DNA often contains secondary metabolites that interfere with analytical applications. In this study, we employed an additional step to remove polysaccharides, polyphenols and secondary metabolites from genomic DNA extracted from young or mature leaf tissue; then a modified traditional cetyl trimethyl ammonium bromide (CTAB) method was applied. The use of 0.4 M glucose improved DNA quality and avoided contamination and browning by polyphenolics, relative to the traditional CTAB method. This is an easy and efficient method for genomic DNA extraction from both young and mature leaves of mango. The isolated DNA was free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by its clear colour, its viscosity, A260/A280 ratio and suitability for PCR-based reactions. This modified protocol was also used to extract high quality genomic DNA from other woody perennials, including walnut, guava, lychee, pear, grape and sugarcane.

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4-6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.

English

An effective method for plant rhizosphere microbial genomic DNA extraction was established. High-purity nucleic acids extracted from soil samples, with considerable yield, could be used for study of soil microorganism molecular ecology. Genomic DNAs were extracted from 12 soil samples of different crop roots. Above 10 &mu;g of genomic DNA with approximately 20-kb fragment length was isolated from 1 g of soil sample. The average value of A260/A230 of the genomic DNA was 1.505, while that of A260/A280 was 1.780. The absorption curves of the full wavelengths of the DNA extracted were consistent with that of pure nucleic acid. DNAs from different dilutions of pollutants were applicable for restriction enzyme digestion analysis, and the lambda DNA in 100-fold diluted soil samples had the same restriction enzyme digestion results as done in ddH2O. Amplicons was produced with the expected molecular size by polymerase chain reaction (PCR).Therefore, the DNA extracted by this method was suitable to be used in analysis of bacterial ecology composition by denaturing gradient gel electrophoresis. &nbsp; Key words:&nbsp;DNA extraction, DNA quality, PCR amplification, soil microbial community diversity, DGGE.

Extraction of nuclear DNA from rhinoceros horn and characterization of DNA profiling systems for white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros

Rhinoceros horn is now worth more, per unit weight, than gold, diamonds, or cocaine. Rhinoceros horn has been used in traditional Asian medicine as a presumed cure for a wide range of ailments. Rhinoceros poaching in South Africa has, on average, more than doubled each year over the past 5 years with the rapid economic growth in east and southeast Asia being assumed to be the primary factor driving the increased demand for horn. Here we report on the characterization of methods for genomic DNA extraction from rhinoceros horn and on DNA profiling systems for white (Ceratotherium simum) and black (Diceros bicornis) rhinoceros. The DNA profiling system described includes 22 short tandem repeat (STR), or microsatellite, markers and a gender marker (ZF1), which have been used previously in various studies on rhinoceros. Using a θ value of 0.1, a conservative estimate of random match probability in 5 white rhinoceros ranged from 1:7.3x10(6) to 1:3.0x10(8). Given that the total population of white rhinoceros is approximately 20,000 such random match probabilities indicate that the genotyping system described provides data which can be used for evidentiary purposes. Furthermore, the methods are appropriate for use in investigations involving trace amounts of rhinoceros horn and the matching of profiles obtained from seized rhinoceros horn with material collected from live animals or poached carcasses.

A rapid and inexpensive one-tube genomic DNA extraction method from Agrobacterium tumefaciens.

Many methods have been used to isolate genomic DNA, but some of them are time-consuming and costly, especially when extracting a large number of samples. Here we described an easy protocol using two simple solutions for DNA extraction from A. tumefaciens cells. Compared with the standard protocol, this protocol allows rapid DNA isolation with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) gDNA extraction was achieved within 15 min; (2) this method was cost-effective, since it only used calcium chloride and lysozyme; SDS, phenol, chloroform and proteinase K were not necessary; (3) the method gave high yield of gDNA (130 ng/loopful culture) compared with standard protocol that was suitable for restriction analysis; (4) the protocol can be carried out in a single test tube and the cells directly from solid media can be used. Thus, this protocol offers an easy, efficient and economical way to extract genomic DNA from A. tumefaciens.

A novel method of genomic DNA extraction for Cactaceae.

• Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated.• Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines.• Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies.