DNA Isolation Protocol Research Articles

AUTENTIKASI DAGING AYAM SEGAR DARI KONTAMINASI DAGING BABI MENGGUNAKAN GEN CYT-B DENGAN ANALISIS DUPLEX- POLYMERASE CHAIN REACTION

Halal is one of important aspects in consumer protection. Meat and processed meat products are food that should be controlled strictly because those are prone to be adulterated by pork contamination. Therefore, it is necessary to provide detection technique which is accurate, fast and cheap. The objective of this research was to identify the presence of impurities of pork meat on raw chicken meat using gene Cyt-b with duplex-PCR analysis. This research used six samples of raw chicken meat and raw pork. Raw chicken meat was bought from supermarkets in the city of Surakarta and raw pork was obtained from pig slaughterhouse. The percentage of raw pork contamination on raw chicken meat was designed as much as 1, 5, 10, and 25%, respectively. The DNA genome was isolated according to DNA isolation protocol from Genomic DNA Mini Kit. In addition, duplex-PCR was performed based on protocol of KAPA2G Fast Multiplex PCR kit. The data was descriptively analyzed by directly looking the DNA bands on the gel documentation apparatus. The result showed that specific DNA bands for chicken and pig were completely appeared on 1.5% of agarose gels. Duplex-PCR detect contamination of pork on raw meat of chicken at all contamination levels. This research proved that the duplex-PCR detect the contamination of pork until the level of 1%.

Optimization of high-yielding protocol for DNA extraction from the forest rhizosphere microbes

Soil is major reservoir for microbes and harbors a vast microbial diversity. Soil microbiota plays a pivotal role in biogeochemical cycles, bioremediation, and in health and disease states of humans, animals, and plants. It is imperative to understand the microbial signatures which are specific in such an ecosystem to unravel their potential role and impact on environment. During the recent years, exploration of soil microbial communities has been geared up with the advent of advanced sequencing technologies. Introduction of custom-made protocols and optimized procedures have enhanced the accuracy levels along with cost-effectiveness of DNA extraction. Standardization of DNA extraction method from soil microbiota has its own limitations due to different nature of soils and the complexity of ecosystems. Though a few standardized protocols are in usage, huge variations and complexities among the microbial communities frequently suggest the optimization, based on various known and unknown factors. Therefore, a set of four standardized DNA isolation protocols was comparatively analyzed with respect to our custom-made protocol owing to the scientific fact that the same protocol does not hold good for all soil samples. Furthermore, the developed protocol has been successfully applied for the identification of efficient plant-specific Rhizobial stains for five legume plants from the soils of various locations under same geographical region. Out of 40 Badrachalam forest soils, five samples, KPFS36, CHFS17, TPFS33, GVFS06, and GPFS40, one for each of Arachis hypogaea, Vigna radiata, Vigna mungo, Glycine max, and Cicer arietinum plants, were selected, respectively, for the soil DNA extraction. A considerable improvement in the DNA yield was identified using the modified protocol with a yield of 21.08 μg/g providing abundant DNA fragments for further investigation on Rhizobial species.

English

The advent and application of molecular markers technology enabled dissection of genomic regions underling important agronomic traits in crop plants. Isolation of sufficient quantity and good quality DNA from a large number of plant samples is a major challenge in most genetic and genomic analyses. Wheat is the world’s third most important food security crop on which most molecular breeding and genetic engineering researches have been undertaken. So far, several wheat DNA isolation protocols and commercial kits are available. However, in some, the cost is so prohibitive for small laboratories, and hence finding cost effective alternative makes small biotechnology laboratories of developing countries beneficial to the technology. The present study was targeted to provide alternative economical, reliable and safer genomic DNA isolation method in wheat. Accordingly, genomic DNA was isolated from 27 bread wheat recombinant inbred lines (RIL) raised in greenhouse with and without liquid nitrogen. From 100 mg, 20-day old leaf samples, averagely 1890.7 ng/μl sufficiently pure genomic DNA (A260/A280 ratio of 1.8) was extracted. In the extraction process, ammonium acetate and sodium acetate solutions were also avoided. Polymerase chain reaction (PCR) analysis of the extracted DNA using two simple sequence repeats (SSR) markers produced polymorphic bands confirming the suitability of the extracted DNA for PCR. Therefore, it is possible to deduce that we have adopted a simple, cost effective, reliable and safer DNA isolation method that can help to extract good quality and sufficiently pure genomic DNA for further molecular studies. Key words: Wheat, genomic DNA isolation, liquid nitrogen, polymerase chain reaction (PCR) analysis, simple sequence repeats (SSR) markers.

Detection of PCR inhibition in food and feed with a synthetic plasmid

We present a successful use of the plasmid inhibition detection and DNA isolation protocol optimisation for four food/feed samples in qPCR analysis of the sequence coding for chloroplast tRNA-Leu: two meat meal samples and two samples made of cranberries (jam and dried fruit). The quantitative real-time polymerase chain reaction (qPCR) can be inhibited by various substances and the DNA content in the sample can be underestimated. It is necessary to identify the PCR inhibition and choose an optimal DNA isolation protocol to correctly evaluate the sample. In a previous study, we have developed an assay using plasmid DNA carrying a non-homologous random sequence identifying possible inhibitors in qPCR in food/feed samples. The plasmid assay allowed to effectively reveal the PCR inhibition in all of the different sample matrices and to choose an optimal DNA isolation protocol.

Standardization of Protocol for Isolation of High Quality Genomic DNA from Polyphenol and Polysaccharide Containing Pyrus Species

Standardization of Protocol for Isolation of High Quality Genomic DNA from Polyphenol and Polysaccharide Containing Pyrus Species

Optimization of DNA extraction protocol in various varieties of papaya for genotyping and molecular diagnosis

Carica papaya L. is a polygamous diploid fruit crop originated from tropical and subtropical regions. Papaya fruit is a good source of vitamin A, B, C (Purnima and Sandhya, 1988), important proteolytic enzyme such as papain and chymopapain with several commercial applications. In tropical plants, higher concentration of RNA, polyphenols, polysaccharides etc, complex the separation and purification of DNA (Qi-Xing Huang et al., 2013). The objectives of the present study were to optimize DNA isolation protocol for 13 different papaya varieties and selectively amplifying the genomic DNA for Polymerase chain reactions. In papaya DNA extraction, there is a problem of browning of pellet, RNA and lipid contamination which decreases yield and quantity. The changes in sample weight, conc. of â-mercaptoethanol, RNAse addition in extraction buffer and no use of liquid nitrogen eliminated contamination and also save time taken for extraction. The DNA A260/280 ratio for varieties were in range between l.6-2.1. Genomic DNA concentration in Pusa nanha was maximum out of 13 varieties. An efficient and fast DNA extraction protocol has been developed without using liquid nitrogen for inter simple sequence repeat (ISSR) and sequence characterized amplified region (SCAR) analysis. The resulted purified genomic DNA showed intact bands in all papaya varieties and was successfully amplified by PCR for several molecular analysis such as, sex determination, diversity analysis, genetic fidielity testing etc.

POLYMORPHISM OF SIMPLE SEQUENCE REPEAT REGIONS OF SULAWESI EBONY (DIOSPHYROS CELEBICA BAKH.) IN EXPERIMENTAL FOREST OF HASANUDDIN UNIVERSITY PROVENANCE

Polymerase Chain Reaction (PCR)-based molecular techniques have been used to detect the polymorphism in plants. The utilization of molecular markers plays essential role in germplasm characterization and plant breeding since the information of DNA marker technology can be exchanged between laboratories and should have standard method to be reproducible. The molecular aspect has been commonly linked to DNA isolation protocol and polymorphic molecular marker, thus can be used for molecular research recommendation purposes. The objectives of this study were to evaluate the capability of microsatellite marker of Ebenaceae Family for amplifying Ebony DNA, and to determine the appropriate PCR annealing temperatures. The DNA isolation of Ebony leaves from Experimental Forest of Hasanuddin University Provenance was carried out using Genomic DNA Mini Kit (Plant) Geneaid protocol. Nine of seventeen selected primers from the Genus Diospyros were able to amplify Ebony DNA. Amplification products produced polymorphic bands with different annealing temperatures (ranged from 53 to 56°C). These nine polymorphic primers will be recommended to use for future studies in genetic diversity as well as pollen dispersal pattern analyses.

Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification

DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120–150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced.Graphical After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr+][Ntf2-]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH2O double-distilled water, qPCR quantitative PCR

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

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

Very Short Mitochondrial DNA Fragments and Heteroplasmy in Human Plasma.

Cell free DNA (cfDNA) has received increasing attention and has been studied in a broad range of clinical conditions. However, few studies have focused on mitochondrial DNA (mtDNA) in the cell free form. We optimized DNA isolation and sequencing library preparation protocols to better retain short DNA fragments from plasma, and applied these optimized methods to plasma samples from patients with sepsis. Our methods can retain substantially shorter DNA, resulting in an average of 11.5 fold increase in short DNA fragments yield (DNA <100bp). We report that cf-mtDNA in plasma is highly enriched in short-size cfDNA (30~60 bp). Motivated by this unique size distribution, we size-selected short cfDNA, which further increased the mtDNA recovery rate by an average of 10.4 fold. We then detected mtDNA heteroplasmy in plasma from 3 patients. In one patient who previously received bone marrow transplantation, different minor allele frequencies were observed between plasma and leukocytes at heteroplasmic sites, consistent with mixed-tissue origin for cfDNA. For the other two patients, the heteroplasmy pattern is also different between plasma and leukocyte. Our study shed new lights into the architecture of the cfDNA, and mtDNA heteroplasmy identified in plasma provides new potential for biomarker discovery.

Genomic DNA Isolation from High Polyphenolic Content Grewia asiatica L. Leaf Without Using Liquid Nitrogen

Grewia asiatica (Grewoideae family) is also known as Phalsa, used for the medicinal purpose has a high content of polysaccharides and polyphenols together with flavonoids and other secondary metabolites which obstructs their pure genomic DNA extraction. Although, there are various good quality DNA extraction protocols, extracting DNA from a high phenolic content plant is a challenging task. The present study describes an efficient and rapid DNA isolation protocol by optimizing cetyl trimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP) and sodium dodecylsulphate (SDS) cocktail specifically for the extraction of DNA from polyphenolic leaf of G. asiatica without using liquid nitrogen. Extracted genomic DNA has high purity index (1.95) examined by A260/A280 ratio. The presence of good quality DNA was also evident by gel electrophoresis. The optimized DNA extraction method is efficient for both fresh and old tough leaves with high polyphenolic contents. The achievement of this method in extracting high quality of genomic DNA demonstrated the broad applicability of this method.

The Miraprep: A Protocol that Uses a Miniprep Kit and Provides Maxiprep Yields.

Plasmid purification is a basic tool of molecular biologists. Although the development of plasmid isolation kits utilizing silica spin columns reduced the time and labor spent on plasmid purification, achieving large plasmid DNA yields still requires significant time and effort. Here we introduce the Miraprep, a rapid protocol that allows isolation of plasmid DNA using commercial Miniprep kits, but with DNA yields comparable to commercial Maxiprep plasmid purifications. Combining ethanol precipitation with spin column purification, we created a DNA isolation protocol that yields highly concentrated plasmid DNA samples in less than 30 minutes. We show that Miraprep isolated plasmids are as stable as plasmids isolated by standard procedures, can be used for standard molecular biology procedures including DNA sequencing, and can be efficiently transfected into mammalian cells. This new plasmid DNA isolation protocol will significantly reduce time and labor without increasing costs.

Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies

Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number.

Comparative analysis of five DNA isolation protocols and three drying methods for leaves samples of Nectandra megapotamica (Spreng.) Mez

The aim of the study was to establish a DNA isolation protocol Nectandra megapotamica (Spreng.) Mez., able to obtain samples of high yield and quality for use in genomic analysis. A commercial kit and four classical methods of DNA extraction were tested, including three cetyltrimethylammonium bromide (CTAB)-based and one sodium dodecyl sulfate (SDS)-based methods. Three drying methods for leaves samples were also evaluated including drying at room temperature (RT), in an oven at 40ºC (S40), and in a microwave oven (FMO). The DNA solutions obtained from different types of leaves samples using the five protocols were assessed in terms of cost, execution time, and quality and yield of extracted DNA. The commercial kit did not extract DNA with sufficient quantity or quality for successful PCR reactions. Among the classic methods, only the protocols of Dellaporta and of Khanuja yielded DNA extractions for all three types of foliar samples that resulted in successful PCR reactions and subsequent enzyme restriction assays. Based on the evaluated variables, the most appropriate DNA extraction method for Nectandra megapotamica (Spreng.) Mez. was that of Dellaporta, regardless of the method used to dry the samples. The selected method has a relatively low cost and total execution time. Moreover, the quality and quantity of DNA extracted using this method was sufficient for DNA sequence amplification using PCR reactions and to get restriction fragments.

An efficient and simple CTAB based method for total genomic DNA isolation from low amounts of aquatic plants leaves with a high level of secondary metabolites

An efficient DNA isolation protocol specifically modified to get pure quality DNA required for molecular studieshas been reported in this paper. Some aquatic plants (Potamogeton spp., Ceratophyllum demersum and Myriophyllum spicatum) were used for the study. The protocol developed will be useful in getting high and pure DNA. Instead of using the available DNA extraction kits, this protocol can be used to get pure quality DNA, freefrom proteins and polysaccharide compounds. The absorbance rate A260/A280 was 1.92 ± 0.069 and A260/A230 was 1.73 by spectrophotometer and NanoDrop machines which showed the sample genomic DNA is pure, free from contaminant proteins and polyphenolics/polysaccharides compound. The highest concentration of DNA was 640 ± 340.58 ng/μl when measured at 260 nm. When we run on agarose gel also, the isolated DNA gave a clear and sharp band. Thus, the DNA does not need any additional purification before proceeding for molecular analysis of the isolated DNA samples. This protocol is very simple and economical which will find wide applications in genomic studies of aquatic plants.

An efficient, simple and high throughput protocol for cotton genomic DNA isolation

Cotton is a stubborn plant for genomic DNA isolation due to its high-level of polyphenolics, polysaccharides and secondary metabolites. Genomics and molecular biology studies require high quality and large quantity of DNA. We have standardized an efficient miniprep protocol for cotton genomic DNA isolation, which not only provides higher yield from 800 to 1400 µg of DNA from 200 to 300 mg of fresh leaf tissue but also provide excellent purity. The DNA is amenable to all elementary enzymatic preparations, PCR techniques, Southern blotting and to high end genomic studies. The technique does not require liquid nitrogen, needs small amount of sample, less time, fewer chemicals and one can process up to 100 samples per day. The genomic DNA extracted was good for transgenic event characterization and marker assisted selection.

Molecular characterisation of coconut (Cocos nucifera L.) varieties using ISSR and SSR markers

ABSTRACTThe present study was carried out to standardise a DNA isolation protocol for coconut and to characterize five coconut varieties using 18 inter-simple sequence repeat (ISSR) and 14 simple sequence repeat (SSR) markers. DNA was extracted from tender young leaf samples collected from the fronds of five different trees of each coconut variety. A protocol using 0.095 g ml−1 glucose, 0.025 g ml−1 polyvinylpyrrolidone, 0.0045 g ml−1 sodium bisulphite, 0.0055 g ml−1 sodium dodecyl sulphate, and 50 µl ml−1 sarcosine produced good quality DNA. The average polymorphism percentages revealed using ISSR or SSR markers between the five varieties were 31.9% or 92.9%, respectively. Using ISSR markers, the overall similarity between all five varieties ranged from 0.657 to 0.775, whereas it was 0.037–0.304 using SSR markers. The levels of polymorphism detected using ISSR markers among the five samples each of ‘Banawali’, ‘Gangabondum Green Dwarf’, ‘Pratap’, ‘Konkan Bhatye Coconut Hybrid-I’, and ‘East Coast Tall’ were 23.2%, 24.2%, 25.6%, 27.1%, and 21.2%, respectively. The levels of polymorphism detected using SSR markers among the five samples of the same five varieties were 85.7%, 86.9%, 85.7%, 100%, and 92.9%, respectively. This study indicated that genetic variation existed both between and within samples of each of the five varieties of coconut. SSR markers were superior to ISSR markers. The extent of genetic variation obtained within a variety was not expected, so it is essential to maintain seed purity via artificial pollination.

An Efficient DNA Extraction Method for Lactobacillus casei, a Difficult-to-Lyse Bacterium

Background: There are several protocols to extract DNA from Lactobacillus spp. In the case of L. casei it is harder because of its especial and thick cell wall. Objectives: In this study, nine DNA extraction protocols (by lysozyme treatment) were evaluated and compared in two categories (traditional and kit-based protocols) and an improved method was presented. Materials and Methods: DNA quantity and quality was determined by spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR). Results: The results revealed that the yield of extracted DNA differed by each protocol (5.8 - 17.1 μg/100 μL), but provided appropriate DNA for PCR amplification. The modified protocol offered the best total DNA extraction method when both quality (DNA purity; 1.54 μg) and quantity (DNA yield; 17.1 μg) were considered. Conclusions: We suggest this protocol for effective and inexpensive DNA isolation from L. casei for downstream biological processes such as PCR.

Application of the RAPD and miRNA markers in the genotyping of Silybum marianum (L.) Gaertn.

Application of the RAPD and miRNA markers in the genotyping of Silybum marianum (L.) Gaertn.

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis.

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.