Optimized method for dna extraction and PCR amplification in aroeira tree

Molecular markers are important tools in the characterization of plant genetic diversity and can provide support for conservation strategies for endangered populations. The different molecular techniques involve the evaluation of many individuals; therefore, it is crucial to have fast, efficient, and inexpensive methods for DNA extraction. Given the importance of the Aroeira (Myracrodruon urundeuva Fr. All.) it is pertinent to optimize a protocol that allows the obtainment of intact and pure DNA, aiming to assist conservation strategies for this species that is threatened with extinction. Thus, this study aimed to compare five DNA extraction methods: Dellaporta et al. (1983), Doyle and Doyle (1987) modified, Ferreira and Grattapaglia (1995), Romano and Brasileiro (2015), and Khanuja et al. (1999) and optimize the most efficient protocol for M. urundeuva. The modified DNA extraction protocol proposed by Doyle and Doyle (1987), using 100 mg of leaf tissue and 6 µl of ß-mercaptoethanol was the protocol that presented the sharpest bands after DNA electrophoresis and after the reactions of amplification employing Polymerase Chain Reaction (PCR). Therefore, it is suggested to use the protocol described by Doyle and Doyle (1987) modified for the extraction of DNA from young M. urundeuva leaves to carry out techniques involving molecular markers.

A simple and cost-effective method for DNA extraction suitable for PCR in "sucupira branca"

"Sucupira branca" is a plant found in the Brazilian Cerrado and is adapted to low fertility soils, and its fruit extract has anti-inflammatory, healing, antiulcerogenic, antimicrobial, cercaricidal, leishmanicidal and antioxidant activities. Furthermore, it provides protection against oxidative stress, is a natural biocontrol agent of Aedes aegypti, has very resistant wood, is a melliferous plant and has been used in reforestation programs. The development of conservation strategies is important for maintaining diversity in natural populations of "sucupira branca" since these populations are in the process of genetic erosion. Molecular biology techniques, which are important for characterizing the genetic diversity of plants to develop conservation strategies, require sufficient high-quality genomic deoxyribonucleic acid (DNA). This study aimed to compare five methods to extract DNA from "sucupira branca". The quality and concentration of DNA were revealed by agarose gel electrophoresis, and only the protocols of Dellaporta, Wood and Hicks et al. (1983) and Khanuja et al. (1999) did not result in satisfactory quantities of DNA. When PCR (Polymerase Chain Reaction) was performed with three inter-simple sequence repeat (ISSR) primers, DNA was successfully amplified from extractions performed with the protocols proposed by Doyle and Doyle (1987), Romano and Brasileiro (1998) and Ferreira and Grattapaglia (1995), which are less expensive than commercial purification kits. These protocols resulted in DNA of sufficient quality and quantity after the amplification reactions were performed.

Preservation and maceration of amazon açai leaflet tissue to obtain genomic DNA / Preservação e maceração de tecido de folíolos de açaí da Amazônia para obtenção de DNA genômico

The objective of this study was to test the efficiency of preservation and maceration methods for Euterpe precatoria leaflet tissue to obtain genomic DNA for molecular studies. The leaflets of E. precatoria were collected in an experimental field at Embrapa Acre, Brazil. The study was conducted in a completely randomized design with 10 replicates, in a 12 × 2 factorial structure, with 12 storage treatments (fresh; lyophiliser 3 days; refrigerator 3, 5, and 7 days; silica gel 7, 10, 20, and 30 days; and transport buffer 3, 5, and 7 days) and two leaf tissue maceration methods (liquid nitrogen and the TissueLyser®). Statistically significant differences in the obtained DNA concentration were found between the maceration and storage treatments. The TissueLyser® macerator produced higher DNA concentrations when compared to liquid nitrogen. For the storage treatments, five groups were formed based on DNA concentration when macerated with the TissueLyser® and two groups when macerated with liquid nitrogen. The DNA concentrations ranged from 285.00 ng/µ L (7 days in transport buffer) to 702.00 ng/µ L (30 days in silica gel) when the leaflets were macerated with liquid nitrogen, and from 572.73 ng/µ L (30 days in silica gel) to 2,850.00 ng/µ L (3 days in lyophiliser) using the TissueLyser® macerator. The DNA purity (A260/A280 nm) varied from 1.30 to 1.70 when the leaflets were macerated with liquid nitrogen and from 1.30 to 1.90 with the TissueLyser® macerator. Despite the variations in leaf tissue preservation and DNA concentration, all treatments were effective for DNA isolation and it was possible to amplify genomic regions of microsatellite markers by PCR. It was concluded that leaflets of E. precatoria stored in a lyophiliser and processed with an automatic macerator resulted in satisfactory DNA for molecular studies.

O objetivo deste estudo foi testar a eficiência de métodos de preservação e maceração de tecidos de folíolos de Euterpe precatoria para obter DNA genômico para estudos moleculares. Os folíolos de E. precatoria foram coletados no campo experimental da Embrapa Acre, Brasil. O estudo foi conduzido em delineamento inteiramente casualizado com 10 repetições, em esquema fatorial 12 × 2, com 12 tratamentos de armazenamento (fresco; liofilizado 3 dias; geladeira 3, 5, e 7; sílica gel 7, 10, 20 e 30 dias e tampão de transporte 3, 5 e 7 dias) e dois tipos de maceração do tecido foliar (nitrogênio líquido e TissueLyser®). Para a variável concentração de DNA houve diferença estatística entre os tipos maceração e de armazenamento. O macerador TissueLyser® apresentou maiores concentrações de DNA quando comparado ao nitrogênio líquido.Para os tipos de armazenamento verificou-se formação de cinco grupos quando macerados TissueLyser® e dois grupos quando macerados com nitrogênio líquido. As concentrações de DNA variaram de 285,00 ng/µ L (7 dias em tampão de transporte) a 702,00 ng/µ L (30 dias em sílica gel) quando maceradas com nitrogênio líquido. Quando maceradas com macerador TissueLyser® variaram de 572,73 ng/µ L (30 dias em sílica gel) a 2.850,00 ng/µ L (3 dias em liofilizador). A pureza do DNA (A260/A280 nm) variou de 1,30 a 1,70 quando os folíolos foram macerados com nitrogênio líquido e de 1,30 a 1,90 quando macerados em macerador TissueLyser®. Apesar das variações na conservação e concentração dos tecidos foliares, todos os tratamentos foram eficazes para o isolamento do DNA e amplificaram regiões de marcadores microssatélites. Concluiu-se que folíolos de E. precatoria armazenados em liofilizador e macerados com macerador automático resultaram em DNA satisfatório para estudos moleculares.

Aspergillus niger as the Source of Ochratoxin A of Contaminated Pyrus communis in Taif Market

Fruits are one of the most important agricultural products that supply the body with vitamins and essential minerals elements, but it is contaminated by fungi during the period of growth, harvesting and storage. A. niger is one of the species that grows on the fruit during the period of storage, and secretes mycotoxins especially ochratoxin A. This study was conducted with the purpose of isolating and identifying different strains of A. niger from 20 samples of pear collected from Taif markets and to determine the ability of these strains to produce OTA. It was observed that showed that out of 20 pear samples collected, 19 samples were detected to be contaminated with different strains of A. niger and the strains were able to produce OTA. From 27 isolates of A. niger which was used to test the ability of production OTA, 10 strains only produced OTA. The range of OTA in all strains were 0.18 to 9.5 ppb. Representative 27 strains of ochratoxigenic and non ochratoxigenic black Aspergilli isolated were subjected for detection of ochratoxin biosynthesis genes, by using two sets of primer for two genes involved in ochratoxin biosynthetic pathway. Bands of the fragments of PKS15C-MeT and PKS15KS genes visualized at 998 and 776 bp, respectively. Whereas, the presence of four tested genes is not sufficient marker for differentatin between aflatoxigenic and non aflatoxigenic isolates.

Comparative analysis of metagenomic DNA extraction methods from gut microbiota of zebrafish (Danio rerio) for downstream next-generation sequencing

Zebrafish (Danio rerio) being a well-established model system can provide significant understanding about interactions between gut microbiota and host system. Gut bacterial diversity may be unraveled only by analyzing metagenomic DNA from diverse higher organisms which helps to identify gut microbes beneficial to the host. Isolation and standardization of metagenomic DNA from zebrafish gut are an inconsequential, but demanding process, as most microbes in the gastrointestinal tract are unculturable. A variety of commercial kits and modified protocols is used for bacterial metagenomic DNA extraction. However, only few methods are reported for the effective isolation of community DNA from zebrafish. The present work portrays an augmented method for gut metagenomic DNA isolation from zebrafish gut, focused on obtaining effective yield and purity. Three methods using kit were evaluated, of which the modified kit method was an affordable and feasible method for isolation of metagenomic DNA from zebrafish. The modified protocol can be used for isolating quality DNA which is an important parameter for downstream applications such as polymerase chain reaction, cloning, next-generation sequencing, and others.

Comparison of Two PCR Assays for Trichomonas vaginalis

PCR is known to be the most sensitive method for diagnosing Trichomonas vaginalis infections. This study aimed to compare the sensitivity of a PCR assay for trichomoniasis (HY-PCR) developed in Hanyang University with the use of a Seeplex Ace Detection Kit®, using urine collected from four Korean men with prostatic disease. Overall, HY-PCR was more sensitive than the Seeplex Kit. The use of Chelex 100 is recommended for DNA isolation in order to increase the sensitivity of the PCR test.

A Rapid Microwave Method for Isolation of Genomic DNA and Identification of White Rot Fungi

A rapid, effective and efficient method to identify the innumerable white rot fungal strains is of utmost importance. Mycelia of the unknown as well as know isolates of WRF, after alternative washing with TE buffer and sterile water, were suspended in TE buffer. Fungi in solution were then exposed to microwave. The crude extract contained genomic DNA which was extracted and amplified using ITS primers for further identification. Based on sequencing results the identity of known cultures was confirmed, while the unknown cultures were identified as Clitopilus scyphoides (AGUM004, BankIt2098576 MH172163); Ganoderma rasinaceum (AGUM007, BankIt2098576 MH172163); Schizophyllum sp (KONA001 BankIt2098576 MH172164; AGUM011 BankIt2098576 MH172165 and AGUM021 BankIt2098576 MH172166 respectively), Coprinellus disseminatus (BANG001, BankIt2098576 MH172167) and Lentinus squarrosulus (TAMI004, BankIt2098576 MH172167). The microwave method described for isolating quality DNA of WRF without further purification steps proved a novel method requiring less than ten minutes and minimized the chances of the presence of PCR inhibitors.

Evaluation of Simple and Cost-Effective DNA Preparation and Subsequent PCR Amplification for Clinically Relevant Mycobacteria.

Aim: Slow growth rate in culture renders the traditional isolation, identification, and drug susceptibility testing of clinically important mycobacteria inadequate when there is an urgent need for a precise diagnosis in order to initiate patient treatment. Molecular methods all rely on mycobacterial DNA isolation which in turn has become an essential step of the process. Our study aimed to evaluate DNA isolation protocols from mycobacteria of clinical interest. Methods: Therefore, in order to determine an optimal method we evaluated 8 inexpensive, rapid and easy DNA isolation methods from 30 mycobacterial cultures (10 Mycobacterium tuberculosis and 20 Non-tuberculous Mycobacteria) for subsequent direct detection by PCR. Results: Six of those 8 methods reliably allow the isolation of good DNA yields and quality, the optimal protocol being the one that includes a 1% Triton X-100 lysis solution. Protocols using SDS 1% as a lysis solution did not yield DNA suitable for PCR amplification. Conclusion: Six of the methods we evaluated can easily be implemented in resource limited settings for routine use, potentially contributing to a better management of mycobacterial infections.

Excised radicle tips as a source of genomic DNA for PCR-based genotyping and melting curve analysis in cotton.

Genomic DNA isolation in cotton is complicated because of the presence of secondary metabolites that are inhibitory to PCR amplification. We report here that radicle tips, but not other parts of cotton seedlings, yield high-quality DNA that is readily amenable for PCR. The radicle-tip-excised seedlings retain viability because of the formation of adventitious roots. We demonstrate the utility of this method in distinguishing homozygotes from heterozygotes in a cotton breeding population and in hybrid seed purity testing.

Stool sample storage conditions for the preservation of Giardia intestinalis DNA

Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT), +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR) amplification was performed using primers that target the β-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month.

Molecular identification of bacteria isolated from meat sample.

Contamination of food with bacteria moulds, yeasts, viruses, protozoa or parasites could occur within food premises and result in unfit or unsafe food. Microbial contamination is frequently the result of ignorance, carelessness or negligence by the food operator, or due to lack of space or inappropriate design of food areas. In this work we have identified meat contaminating bacteria through molecular techniques. The bacterium identified through morphological and biochemical tests was sporosarcina halofila. This was confirmed after amplification of genomic DNA with suitable primers. Plasmid was isolated and it was identified to be 11kb. PCR amplified product was 100bp and primers were designed accordingly to the suitable organism.

Methodological variations in the isolation of genomic DNA from Streptococcus bacteria

In this work, genomic DNA of Streptococcus pyogenes, S. mutans and S. sobrinus was isolated using two methods: either using the detergent cetyltrimethylammonium bromide (CTAB) at 65ºC; or by applying ultrasound to a mixture of silica and celite in CTAB. The composite method that used ultrasound was the more efficient, allowing the straightforward extraction of genomic DNA from Gram-positive bacteria with good quality and reproducibility.

O gênero Streptococcus encontra-se amplamente distribuído na natureza e algumas espécies constituem a microbiota humana da cavidade bucal, como Streptococcus pyogenes, que pode estar associado a importantes doenças humanas, Streptococcus mutans e Streptococcus sobrinus, relacionados à cárie dental. O DNA genômico destas três espécies foi isolado utilizando-se dois métodos, o primeiro utilizando o detergente brometo de cetiltrimetilamônio (CTAB) à 65ºC e outro associando ultra-som a uma mistura de sílica e celite em CTAB. O método que possibilitou a extração do DNA genômico das bactérias Gram positivas, com qualidade, boa reprodutibilidade fácil execução foi aquele que utilizou ultra-som associado à sílica e celite em CTAB.

Isolation of genomic DNA from medicinal plants without liquid nitrogen.

Genomic DNA was extracted from eight medicinal plants using the present DNA extraction protocols (CTAB extraction method) with some modifications. Leaves were fixed in different fixing solutions containing absolute alcohol (99.99%), chloroform and EDTA, but without liquid nitrogen. DNA quality and quantity obtained were comparable to those isolated with liquid nitrogen, as the λ260/λ280 ratio with liquid nitrogen was in range 1.3-1.7 and with other fixing solutions it was 1.1-1.5. Absolute alcohol showed best results as fixing solution. Good quality of DNA was isolated without using liquid nitrogen from different medicinal plant species. DNA isolated by this method was suitable for various molecular biology applications.

An alternative method for Staphylococcus aureus DNA isolation / Metodologia alternativa para extração de DNA genômico de Staphylococcus aureus

This study describes a rapid procedure for the isolation of genomic DNA from Staphylococcus aureus that yielded a good amount of high quality DNA for the amplification of staphylococcal enterotoxins genes (A, B, C, D, and E) and the TSST-1 gene as well as enzymatic restriction (HaeIII) from environmental isolates. With this method, it was possible to detect these genes in a sample containing as little as 10(5) cells with positive PCR reactions obtained from approximately 10pg of DNA in a final reaction volume of 25µl.

Descreve-se um procedimento rápido para extração de DNA genômico de isolados de Staphylococcus aureus capaz de produzir DNA estafilocócico em qualidade e quantidade suficiente para a amplificação de genes que codificam enterotoxinas estafilocócicas (A - E) e para TSST-1 e restrição enzimática (HaeIII) de isolados ambientais. O método proposto foi capaz de detectar esses genes em um produto de extração contendo tanto quanto 10(5) células, e reações positivas de PCR foram obtidas de aproximadamente 10pg de DNA.

A phenol-free method for DNA isolation from human blood / 基础医学与临床

Objective To find an ideal method of DNA isolation from blood and especially from clotted blood and to minimize the volume of blood collected for laboratory and clinical tests.Methods DNAs were isolated from antiagglutinated and agglutinated blood samples from auricular veins of 30 healthy subjects. The DNAs of these samples were obtained by a nonenzymatic, nontoxic procedure optimized by us and determinated by agarose gel electrophoesis and PCR. Results The yields of DNA isolated from clotted blood and antiagglutinated blood were (40.2?8.86)mg DNA/L and (39.1?10.2)mg DNA/L, and purities were 1.87?0.11 and 1.92? 0.12. The DNAs that we isolated from all samples had high molecular weight and by PCR the dimorphism of ALU alleles of the 8th intron of t-PA was easy to be obtained, so they were complete and reliable. Conclusion This method is rapid, easy, efficient and nontoxic for isolation of DNA from clotted and fresh blood and meets requirements for clinical testing and molecular biology study.

Optimization of conus genomic DNA isolation methods / 中国海洋药物

Conopeptides (Conotoxin) derived from tropical marine gastropod, cone snail ( Conus ) venoms have been an useful tool agent in neuro investigation and new drug source. Recently, new method of Conotoxin genes cloning from Conus genomic DNA has been established. So to get Conus genomic DNAs rapidly is the basis for diverse Conotoxin gene isolation, gene bank construction and the medical source exploitation. In this report, different DNA extractions from different tissue and organs of six cone snail species were performed in order to optimize the DNA isolation methods. The optimized brief phenol SDS method was established which fit for cone snails genomic DNA extraction.