DNA Isolation Protocol Research Articles

Simplified extraction of good quality genomic DNA from a variety of plant materials

Depending on the nature and complexity of plant material, proper method needs to be employed for extraction of genomic DNA, along with its performance evaluation by different molecular techniques. Here, we optimized and employed a simple genomic DNA isolation protocol suitable for a variety of plant materials covering in vitro grown tender plantlets to relatively complex plant tissues such as field grown mature potato leaves and tubers. Unlike other methods, no detergent was included in the isolation steps. This protocol, based on Dellaporta’s method as reported earlier, worked efficiently both at small and miniscale during handling large number of plant materials. DNA yield was found to be in the range of 70 to 120 mg per gram of the plant material; sufficient for most of the molecular techniques. Purity of DNA was checked by A260/A280 ratio, and restriction analyses including the isoschizomers HpaII and MspI. The DNA preparations were successfully used in polymerase chain reactions using gene-specific primers for cloning of different genes. Prolonged storage did not affect the quality of the DNA samples. Taken together, this method could be a reliable substitute to frequently used chemical cetyl trimethylammonium bromide (CTAB) and commercial kits-based plant DNA isolation protocols. Key words: Plant materials, genomic DNA isolation, restriction analyses, HpaII andMspI isoschizomers, gene-specific primers, polymerase chain reaction (PCR), molecular cloning, DNA storage.

Sensitive life detection strategies for low-biomass environments: optimizing extraction of nucleic acids adsorbing to terrestrial and Mars analogue minerals

The adsorption of nucleic acids to mineral matrixes can result in low extraction yields and negatively influences molecular microbial ecology studies, in particular for low-biomass environments on Earth and Mars. We determined the recovery of nucleic acids from a range of minerals relevant to Earth and Mars. Clay minerals, but also other silicates and nonsilicates, showed very low recovery (< 1%). Consequently, optimization of DNA extraction was directed towards clays. The high temperatures and acidic conditions used in some methods to dissolve mineral matrices proved to destruct DNA. The most efficient method comprised a high phosphate solution (P/EtOH; 1 M phosphate, 15% ethanol buffer at pH 8) introduced at the cell-lysing step in DNA extraction, to promote chemical competition with DNA for adsorption sites. This solution increased DNA yield from clay samples spiked with known quantities of cells up to nearly 100-fold. DNA recovery was also enhanced from several mineral samples retrieved from an aquifer, while maintaining reproducible DGGE profiles. DGGE profiles were obtained for a clay sample for which no profile could be generated with the standard DNA isolation protocol. Mineralogy influenced microbial community composition. The method also proved suitable for the recovery of low molecular weight DNA (< 1.5 kb).

Season, Environment Stress and Refrigerated Storage Affect Genomic DNA Isolation of Tung Tree

Many metabolites in leaf tissue disturbed plant genomic DNA isolation and always varied when leaves was harvested from different environments. Objective of this study was to investigate whether season, environment stress and refrigerated storage affect genomic DNA isolation of tung tree leaves. Five types of young leaves and two DNA isolation protocols, the recycling CTAB protocol I and II, were adopted to carry out the experiment. Our results showed that both leaf type and protocol affected DNA isolation of tung tree. Using the recycling CTAB protocol II, though little DNA were obtained from three types of young leaves, the other two have satisfying results. Whereas the recycling CTAB protocol I could produce high yield genomic DNA from all the five types of young leaves. All the detectable DNA samples in agarose gel electrophoresis were good templates for PCR reaction. Season, environment stress and refrigerated storage had a big effect on genomic DNA isolation of tung tree. The recycling CTAB protocol I was proved to be an effective and universal protocol for DNA isolation of tung tree. Five types of young leaves could all act as the tissue for isolation of genomic DNA, but the summer healthy young leaves without long-time refrigerated storage are the best. The optimal leaf tissue will benefit DNA isolation of plant species.

Comparison between modified DNA extraction protocols and commercial isolation kits in grapevine (Vitis vinifera L.)

Various protocols have been developed and used for DNA extraction in grapevine. However, owing to the long duration of the isolation steps in previously developed protocols, researchers have preferred to use isolation kits for studies in recent years. In our study, the DNA yield and purity obtained using six methods--namely three DNA isolation protocols and three commercial DNA isolation kits--were compared. Modifications were made and the isolation steps were shortened in the previously developed DNA isolation protocols to achieve more rapid and practical protocols. The samples were taken from plants grown under vineyard and greenhouse conditions in two periods during spring and autumn. The best results among the six DNA isolation methods were discussed. The results were also supported with polymerase chain reaction analyses conducted with isolated DNAs.

PCR-compatible genomic DNA isolation from different tissues of rice (Oryza sativa) for SSR fingerprinting

Background: In the genomic era, polymerase chain reaction (PCR) based DNA marker analysis is widely used for several crop plants, especially in rice (Oryza sativa), for several improvemental aspects. Such study requires a fast, inexpensive, and suitable DNA isolation protocol having several overall advantages. The aim of this work was to standardize a DNA isolation protocol for rice which should be simple, cost-effective, high throughput, PCR compatible, and needs a small amount of plant tissues without using liquid nitrogen. Materials and Methods: To fulfill such a desired goal, genomic DNA was isolated from different tissues (seedling, leaf, root, grain, kernel, straw, and embryogenic callus) of the rice plant following a modified protocol. The isolated DNA was subjected to PCR amplification with a reported trait linked rice-microsatellite (RM) marker. Results: The quality and quantity of the isolated genomic DNA from this modified protocol proved to be comparable with the other standardized protocols. The microsatellite based DNA fingerprint shows reproducible bands from different isolated DNA tissue sources. Conclusions: This mini prep cost-effective protocol was standardized with few milligrams of fresh and dried tissues, it does not require liquid nitrogen, can handle large number of samples in a working day per worker, and be efficiently applied to rice. The protocol has now also been applied in other plants like wheat and mungbean yielding about 0.55 μg of high molecular weight DNA from 100 mg of plant material with negligible RNA contamination.

Optimization of Protocol for Isolation of Genomic DNA from Leaves of Selaginella Species Suitable for RAPD Analysis and Study of their Genetic Variation

Abstract A simple and efficient protocol for isolating genomic DNA from leaves of Selaginella spp. (S. delicatula, S. repanda, S. bryopteris, S. plana, S. monospora) was developed, involving a modified CTAB protocol of Rogers and Benedich (1994). Increasing the incubation time with the precipitation buffer (1X CTAB) from 1–3 hours to 12–14 hours helped achieve higher quantity genomic DNA from the specimens, when compared with DNA extracted by protocols reported by Dellaporta et al. (1983), Murray and Thompson (1980) and Doyle and Doyle (1987). The DNA yield ranged from 846–1836 µg/ml from fresh and herbaria-preserved leaf samples. The DNA samples were found suitable for genetic diversity analysis with Random Amplified Polymorphic DNA (RAPD) markers. Nine random primers (OPA A17, OPB 4, OPB13, OPC 2, OPC 11, OPD 5, OPG 2, OPG 19 and OPK 10) were studied, of which two primers (OPD 5 and OPG 2) yielded reproducible amplification profile of polymorphic fragments.

Evaluation of rapid protocols for DNA isolation from Cercospora beticola Sacc.

The most fungal DNA isolation protocols are designed to obtain high amounts of very pure DNA, requiring large fungal cultures and extraction procedures with many purification steps. Since the PCR does not require high purity DNA, the aim of this investigation was to evaluate three fast and simple fungal DNA isolation protocols for further use in Cercospora PCR based research. The purity and quantity of isolated DNAs were determined spectrophotometrically, electrophoretically and by PCR reaction with universal primers. The amounts of DNA evaluated on agarose gels, isolated by protocols A and C, did not correspond to the spectrophotometrical values, probably due to RNA impurities. In samples isolated by protocol B these impurities were not detected and the DNA concentrations were more similar. Neither protocol eliminated impurities such as carbohydrates and phenol. The average DNA yield of protocol A was 1.04 μg/μl, protocol B 0.88 μg/μl, and protocol C 0.55 μg/μl. The DNA quality most suitable for PCR analysis was obtained by protocol A, where amplification product with universal primers was detected in all DNA samples. The amplification product was detected in 87% of samples isolated by protocol C and in only 60% of samples isolated by protocol B. Although DNA obtained by protocol A had the highest yield and best quality, the isolation protocol C should be also recommended, for it does not require phenol, chlorophorm or liquid nitrogen.

OPTIMIZACIÓN DE UN PROTOCOLO DEL AISLAMIENTO DEL ADN Y DE UN SISTEMA DE AMPLIFICACIÓN ISSR-PCR PARA Ceratozamia mexicana Brongn. (Zamiaceae)

La mayoría de las cícadas contienen altas concentraciones de aceites esenciales, flavonoides, polifenoles y polisacáridos que interfieren en la extracción de ADN, causando productos de amplificación errados o inhibiendo la PCR. La optimización del aislamiento del ADN y el empleo de iniciadores de secuencias intergénicas repetidas simples (ISSRs) se investigaron en Ceratozamia mexicana Brongn., una cícada mexicana en peligro de extinción. El ADN obtenido de tejido foliar fresco, con un amortiguador modificado de cetil trimetil amonio, nos permitió obtener un ADN de buena calidad, sin pigmentos coloridos o contaminantes. La modificación al protocolo de extracción de ADN, basado en CTAB, fue un prelavado por 1 h, del tejido foliar, con una solución de 0.7 M de NaCl, para facilitar la lisis celular. El ADN extraído exitosamente se amplificó por PCR, usando seis iniciadores arbitrarios ISSR. Se observaron productos de amplificación reproducibles en todas las reacciones de PCR. Nuestros resultados muestran que la implementación mejora significativamente la calidad del ADN obtenido, usando una concentración baja de iniciadores (25 pM). Se detectaron 23 bandas fuertes, nueve de las cuales fueron polimórficas. Los resultados indican que el protocolo de optimización del aislamiento del ADN y en el sistema de PCR es viable para futuros trabajos en esta especie. Este trabajo es el primer protocolo de extracción de ADN y de ISSR reportado para esta especie ornamental en peligro de extinción.

Development of genomic tools for verification of hybrids and selfed progenies in cassava (Manihot esculenta)

Cross contamination arising from random natural processes and breeding errors is one of the prominent challenges to breeding in many crops including cassava ( Manihot esculenta ). This study attempts to identify molecular marker tools suitable for verifying the genetic purity of putative progenies of populations of cassava. It also proposes a rapid, high-throughput genomic DNA extraction protocol which is a modification of an existing extraction protocol adapted to the pace required for the DNA-based verification of often large population sizes. Three polymorphic simple sequence repeat (SSR) markers were selected from a total of 125 and used for genotyping three populations. The petiole color trait was also used to verify TMS 96/1089A X TME117 where the pink color of the male parent was dominant over the female’s green color. The pace of genomic analysis of populations used in the study was enhanced using a modified , quicker DNA isolation protocol which slashed extraction time by 60%. SSRY153 identified six false progenies in the selfed line, 1M18. Segregation patterns of In combination, both markers identified 2 selfed individuals out of a total of 207. NS890 identified two false hprogenies resulting from selfing in the female parent of the cross TMS 30001 X TMS 96/1089A, out of a total of 93, The principles described for verification using each of the SSR markers applied in this study can be extended to other SSR markers with corresponding nature of polymorphism in any population of the crop. Increased attention would go to the development of more efficient markers such as single nucleotide polymorphisms (SNPs) and other gene-based markers using new and advanced genomics techniques for routine integration of such quality control step in the breeding scheme. Key words: Cassava, simple sequence repeat (SSR), morphological trait, molecular markers, genomic DNA.

A Rapid and Efficient Method for Isolating High Quality DNA from Leaves of Carnivorous Plants from the Drosera Genus

Drosera rotundifolia, Drosera capensis, and Drosera regia are carnivorous plants of the sundew family, characterized by the presence of stalked and sticky glands on the upper leaf surface, to attract, trap, and digest insects. These plants contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites that interfere with DNA isolation and subsequent enzymatic reactions such as PCR amplification. We present here a protocol for quick isolation of Drosera DNA with high yield and a high level of purity, by combining a borate extraction buffer with a commercial DNA extraction kit, and a proteinase K treatment during extraction. The yield of genomic DNA is from 13.36 μg/g of fresh weight to 35.29 μg/g depending of the species of Drosera, with a A₂₆₀/A₂₈₀ ratio of 1.43-1.92. Moreover, the procedure is quick and can be completed in 2.5 h.

Standardization of DNA Isolation and PCR Protocol for RAPD Analysis of Suaeda sp.

Standardization of DNA Isolation and PCR Protocol for RAPD Analysis of Suaeda sp.

Buffy coat specimens remain viable as a DNA source for highly multiplexed genome-wide genetic tests after long term storage

BackgroundBlood specimen collection at an early study visit is often included in observational studies or clinical trials for analysis of secondary outcome biomarkers. A common protocol is to store buffy coat specimens for future DNA isolation and these may remain in frozen storage for many years. It is uncertain if the DNA remains suitable for modern genome wide association (GWA) genotyping.MethodsWe isolated DNA from 120 Action to Control Cardiovascular Risk in Diabetes (ACCORD) clinical trial buffy coats sampling a range of storage times up to 9 years and other factors that could influence DNA yield. We performed TaqMan SNP and GWA genotyping to test whether the DNA retained integrity for high quality genetic analysis.ResultsWe tested two QIAGEN automated protocols for DNA isolation, preferring the Compromised Blood Protocol despite similar yields. We isolated DNA from all 120 specimens (yield range 1.1-312 ug per 8.5 ml ACD tube of whole blood) with only 3/120 samples yielding < 10 ug DNA. Age of participant at blood draw was negatively associated with yield (mean change -2.1 ug/year). DNA quality was very good based on gel electrophoresis QC, TaqMan genotyping of 6 SNPs (genotyping no-call rate 1.1% in 702 genotypes), and excellent quality GWA genotyping data (maximum per sample genotype missing rate 0.64%).ConclusionsWhen collected as a long term clinical trial or biobank specimen for DNA, buffy coats can be stored for up to 9 years in a -80degC frozen state and still produce high yields of DNA suitable for GWA analysis and other genetic testing.Trial RegistrationThe Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial is registered with ClinicalTrials.gov, number NCT00000620.

A first report on isolation of DNA in Bryocladia thwaitesii (Harvey ex J. Agardh) Detoni (red alga) using Nano vue UV spectrophotometer suitable for molecular biological studies

Bryocladia thwaitesii (Harvey ex J. Agardh) Detoni, is a commercially important red algae finding wide use in pharmaceutical, cosmetics, nutraceutical industries. However there is no work was made on the DNA isolation which is the key step for further improvement of its yield. Here, for the first time we report the DNA isolation suitable for molecular biological studies and the quality and quantity of the DNA is reported in comparison with the green alga ( Ulva covelongenesis V. Krishnamoorthy and H.Joshi). Optimization of DNA isolation was done in these algae by adopting different methods. In the optimization process, different DNA isolation methods were followed. A better result in terms of purity and yield was obtained in method suggested by Wattier et al . (2000) modification by Dellaporta et al. (1983). Quantitative analysis made using Nano vue UV spectrophotometer sets as a pioneering effort in the B. thwaitesii . The DNA isolated by this protocol from two seaweeds, both the purity of DNA as well as concentration were very high i.e., 1.772 and 594.5 mg/μl respectively in B. thwaitesii . In U. covelongenesis the purity of DNA was 1.763 and concentration of DNA 569.5 mg/μl, which was fairly high in other protocols studied, the purity of DNA ranged from 1.444 to 1.640 and concentration of DNA ranged from 71 to 463 ngm/μl. All the experiments were performed by keeping the green alga U. covelongenesis as standard for comparison. The DNA isolation protocol indentified in the present investigation in the seaweed B. thwaitesii would become well suited for PCR amplification and genomic library construction.

A First Report on Isolation of DNA in Bryocladia thwaitesii (Harvey Ex J. Agardh) Detoni (red Alga) Using Nano Vue Uv Spectrophotometer Suitable for Molecular Biological Studies

Bryocladia thwaitesii (Harvey ex J. Agardh) Detoni, is a commercially important red algae finding wide use in pharmaceutical, cosmetics, nutraceutical industries. However there is no work was made on the DNA isolation which is the key step for further improvement of its yield. Here, for the first time we report the DNA isolation suitable for molecular biological studies and the quality and quantity of the DNA is reported in comparison with the green alga ( Ulva covelongenesis V. Krishnamoorthy and H.Joshi). Optimization of DNA isolation was done in these algae by adopting different methods. In the optimization process, different DNA isolation methods were followed. A better result in terms of purity and yield was obtained in method suggested by Wattier et al . (2000) modification by Dellaporta et al. (1983). Quantitative analysis made using Nano vue UV spectrophotometer sets as a pioneering effort in the B. thwaitesii . The DNA isolated by this protocol from two seaweeds, both the purity of DNA as well as concentration were very high i.e., 1.772 and 594.5 mg/μl respectively in B. thwaitesii . In U. covelongenesis the purity of DNA was 1.763 and concentration of DNA 569.5 mg/μl, which was fairly high in other protocols studied, the purity of DNA ranged from 1.444 to 1.640 and concentration of DNA ranged from 71 to 463 ngm/μl. All the experiments were performed by keeping the green alga U. covelongenesis as standard for comparison. The DNA isolation protocol indentified in the present investigation in the seaweed B. thwaitesii would become well suited for PCR amplification and genomic library construction.

A DNA Isolation Protocol Suitable for RAPD Analysis from Fresh or Herbarium-Stored Leaves of a Historic Quercus virginiana L.

A DNA Isolation Protocol Suitable for RAPD Analysis from Fresh or Herbarium-Stored Leaves of a Historic Quercus virginiana L.

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。

Easy and efficient protocol for oomycete DNA extraction suitable for population genetic analysis

Purpose of workA simple and rapid DNA extraction protocol capable of obtaining high-quality and quantity DNA from a large number of individuals is essential for assaying population and phylogenetic studies of plant pathogens. Most DNA extraction protocols used with oomycetes are relatively lengthy and cumbersome for high throughput analysis. Commercial kits are widely used, but low quantities of DNA are usually obtained, and with large scale analysis multiple isolations are required.A protocol for DNA isolation from Phytophthora and Pythium suitable for the evaluation of a large set of molecular markers was modified from one previously developed for soybean seed. There was a one to three fold increase in the amount of DNA that was extracted using the modified protocol compared to a commercial kit. The DNA obtained using the modified protocol was suitable for the amplification of microsatellite markers as well as the ITS region. This protocol is inexpensive, easy, quick, and efficient in terms of the volume of reagents and the number of steps involved in the procedure. The method may be applicable to other oomycetes and effectively implemented in other laboratories.

Isolation of Plant DNA for PCR and Genotyping Using Organic Extraction and CTAB

A general difficulty in isolation of DNA from plant cells is the presence of a cell wall. It is necessary to degrade plant cell walls, either physically or enzymatically, in order to effectively isolate plant DNA. Additionally, some tissues (such as endosperm) or some species contain high levels of starches or phenolic compounds that can complicate DNA isolation. A number of plant DNA isolation protocols are designed to overcome species-specific difficulties. This is a relatively simple protocol that uses an extraction buffer containing cetyltrimethylammonium bromide (CTAB); it can be used for many plant species. It provides a substantial amount of high-quality DNA that is suitable for polymerase chain reaction (PCR) procedures and is stable for long periods of time. The cost per sample is very low. In addition, this protocol is relatively robust and can be performed by individuals who have had relatively little training. A typical undergraduate student can perform ~200-300 isolations in a day using this protocol. The disadvantages are that it requires a freeze-dryer and a mill or paint-shaker-like device and that it utilizes an organic extraction step, requiring the use of a fume hood.

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.

Recipes for Life Scientists

BioTechniquesVol. 49, No. 5 From the EditorOpen AccessRecipes for Life ScientistsNathan S. BlowNathan S. Blow*E-mail Address: nathan.blow@informausa.comBioTechniquesSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000113531AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Starting in a new lab back in 1993, I was given a single sheet of paper describing an approach for isolating DNA from gel fragments. It was a Benchmark article published in this journal the year before, with several post-it notes added to the front from various lab members describing critical stopping points and conditions. Upon photocopying, the post-it notes became a permanent record of how to best isolate the DNA. To my delight, the approach worked amazingly well, saving both time and money. For the next ten years, this single sheet of paper would follow me from lab to lab, with copies often being left with other scientists who were searching for a better way to isolate DNA for cloning.This was my first introduction to the fact that detailed protocols derived from methods articles can be as valuable a tool in the lab as any piece of equipment or kit. The DNA isolation Benchmark was not a protocol at first; the post-it notes and handwritten instructions added to the page created a “community” protocol over the years. What protocols provide researchers is a series of steps and guidelines to stick with while performing experiments. As a methods journal, we strive to assist our authors in communicating their techniques and methods as clearly as possible, enabling readers to use these new approaches successfully in their own research.For several years now, to accompany our main issue, BioTechniques has been publishing an annual Protocol Guide at the end of the year. In the past, this guide featured a wide variety of protocols from different sources. This year, with the DNA isolation story in mind, we wanted to take our annual protocol guide a step farther, providing readers with a series of detailed protocols from some of our most popular articles published during 2010. While providing readers insights into various approaches and techniques, we sought to give readers the necessary advice and information to make these techniques and methods even more effective in their labs. To this end, this year you will find hints and attention steps that point out places where one can stop and rest, or specific steps where special attention should be paid. In addition, several protocols this year include online troubleshooting sections where researchers can look if something does go amiss during the experiment. It is our hope that with all this additional information and detail, the protocols in this year's guide will become a crucial piece of life in the lab much the way the DNA isolation protocol was for my labmates and me.This is the first year in which such detailed protocols are being presented in our guide, and we would like to encourage future authors of BioTechniques articles to consider supplying protocols with their manuscripts as supplementary materials when warranted. Such protocols will serve to enhance the visibility of a method or technique, giving scientists the opportunity to further expand their research efforts by more quickly adapting a method for their use. If you have a protocol story or would like to comment on one of our current protocols, please share your thoughts with us by posting at our Molecular Biology Forums under “To the Editor” (http://molecularbiology.forums.biotechniques.com) or sending an email directly to the editors (bioeditor@biotechniques.com).FiguresReferencesRelatedDetails Vol. 49, No. 5 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print November 2010 Information© 2010 Author(s)PDF download