Isolation Of Total DNA Research Articles

Novel kit for efficient extraction of long-segment genomic DNA by nanodisk adsorption

The acquisition of complete DNA is usually the first and most critical step in many basic molecular biology applications. Isolation of complete total DNA from tissue samples is related to different physical and biochemical properties of tissue. Long fragments of DNA are easily degraded and fragmented, which poses a challenge for extracting complete long fragments of DNA. By comparing several different extraction methods and verifying the concentration and length of extracted DNA, it is proved that the kit we used has high efficacy. We provide tissue kit DNA extraction for long fragments and obtain long fragments of high purity DNA from almost any type of tissue, especially muscle and blood tissues.

Molecular characterization and haplotypes of sheep and goat isolates of Cysticercus tenuicollis in Turkey.

Taenia hydatigena, is a common parasite, lived mostly in dogs and wild carnivores in its mature stage, and the larvae, Cysticercus tenuicollis, is found on ruminants and pigs. The aim of the current study was to determine the genetic diversity in 20 isolates of the sheep and goats. After the isolation of total genomic DNA from C. tenuicollis isolates, genetic characterization of the mitochondrial NADH dehydrogenase subunit 1 gene region was amplified using specific JB11-JB12 primers in PCR and the PCR products were sequenced and haplotype and genetic diversity analyses were utilized. As a result, multiple nucleotide changes were determined in the sequence analyses of the isolates leading to detection of 16 and 15 different haplotypes in sheep and goat samples, respectively. These findings are important in terms of showing the diversity of nucleotide variation in C. tenuicollis in Turkey.

Amplification and Analysis of Rbcl Gene (Ribulose-1,5-Bisphosphate Carboxylase) of Clove in Ternate Island

DNA Barcoding is recommended as a tool for identifying and confirming species within the taxonomy framework. The rbcL gene is the barcode DNA for plant species. Ternate Island is one of clove plantation center in North Maluku. The diversity and productivity of cloves on the Ternate island known since in earlier times. This study has a purpose to amplify the rbcL gene in clove plants collected from clove plantations of communities in Ternate island. Isolation of total DNA carried out with using ZymoBiomic (Zymo Research DNA Extraction) Kit from leaves tissue, then DNA specimen is amplified based on rbcL gene with forwarding sequence rbcLaF 5′-ATG TCA CCA CAA ACA GAG ACT AAA GC-3′ and reverse sequence is rbcLaR 5′-GTA AAA TCA AGT CCA CCR CG-3′. The results showed that the specimen was successfully amplified with an amplicon size of 600 bp. Furthermore, BLASTN analysis results note that the sequence has a similarity of 99% with Syzygium cumini vc. J.R. Abbott 23676 (FLAS) ribulose-1,5-bisphosphate carboxylase large subunit (rbcL) gene, but phylogenetically the precise position of sample could not found because the limitation of rbcL gene.

Genetic Diversity Analysis of Aydın/Turkey Dittrichia viscosa (L). Greuter (Asteraceae) Populations Using RAPD Markers

In this study, we performed a genetic diversity using RAPD markers for some Dittrichia viscosa populations grown in the Aydın region of Turkey. Total genomic DNA isolation from the leaves of Dittrichia viscosa was performed using commercial kit. eight RAPD primers were used to determine genetic diversity among populations. Polymerase Chain Reaction (PCR) was performed with all DNA samples and primers with ability of scoreing band. PCR products were run in agarose gel and visualized under UV light. They scored as there were bands (1) and no bands (0) at all gel images and their files were created. A total of 50 characters were obtained from the primers. Phylogenetic relationships and genetic distances between the cultivars were calculated by using the PAUP* 4.0b10 analysis program. According to the PAUP data, the closest genetic distance was determined 0.20000 and between Central and İncirliova populations, the most distant genetic distance were determined 0.36842 between İncirliova and Koçarlı populations. In the phylogenetic analysis has been obtained using UPGMA algorithms and phylogenetic tree consist of two clades. The results also propose that RAPD markers are useful tools for indicating genetic relationships among Dittrichia viscosa populations.

The Impact of Dietary Grape Seed Meal on Healthy and Aflatoxin B1 Afflicted Microbiota of Pigs after Weaning.

The study investigated the effect of grape seed (GS) meal, aflatoxin (AFB1), or their combination on the large intestine microbiota of weanling piglets. Twenty-four piglets were allocated into four groups based on diet composition: (1) Control group; (2) AFB1 (320 g/kg feed) group; (3) GS group (8% inclusion in the diet); (4) AFB1 + GS group. After 30 days of experiment, the colon content was used for microbiota analyses; after isolation of total bacterial genomic DNA, V3/V4 regions of the 16S rRNA amplicons were sequenced using the Illumina MiSeq platform. The raw sequences were analyzed using the v.1.9.1 QIIME pipeline software. 157 numbers of OTUs were identified among all four dietary groups with 26 of them being prevalent above 0.05% in the total relative abundance. GS and AFB1 increase the relative abundance of phylum Bacteroidetes and Proteobacteria, while decreasing the Firmicutes abundance in a synergic manner as compared with the individual treatments. An additive or synergistic action of the two treatments was identified for Lactobacillus, Prevotella and Campylobacter, while rather an antagonistic effect was observed on Lachnospira. The action mechanisms of aflatoxin B1 and grape seed meal that drive the large intestine microbiota to these changes are not known and need further investigations.

A cost-effective protocol for total DNA isolation from animal tissue

ABSTRACTLaboratories that perform PCR on a routine basis need to count on reliable DNA isolation methods. In locations where supply of DNA extraction kits depends on importation, having an in-house protocol is desirable. This is also important for laboratories limited by budget constraints. We present a low-cost DNA isolation protocol that incorporates well-known techniques, but that we have adapted to various animal tissues. We tested this protocol on animal blood and muscle, and on cell suspension from skin swabs. The results were comparable, in terms of amount and quality of DNA, to those obtained with two other commercially available methods. DNA retrieved with this protocol has been successfully employed for Sanger sequencing of gene PCR products from animal tissues and blood, as well as for PCR-based diagnosis of chytrid fungus in amphibians and blood parasites in birds.

The application of barcode DNA rbcL gene for identification of medicinal plants: red jabon and gofasa

Red jabon and gofasa plants has potential as a medicinal plant. One of the potential drugs of these two plants as anti-cholesterol purpose of this study was to identify the red jabon and gofasa plants in Ternate Island using rbcL gene of DNA barcode. The method in this study consists of total DNA isolation using ZymoBiomic (Zymo Research DNA Extraction) Kit, DNA amplification using rbcL gene, sequencing and BLASTn analysis. Based on barcode DNA rbcL, the red jabon plants has similarities 99% Cephalanthus occidentalis, Mitragyna speciosa and Ocherreuclear sp. for gofasa plants has 99% resemblance with Vitex glabrata, Vitex megapotamica, Vitex trifolia and Vitex negundo at NCBI database. The application of rbcL barcode DNA is effective to identify plants at the family level up to the genus.

Genetic Diversity Based on ISSR Markers of Apple Genotypes in Ardahan/Turkey

Within the present study, it was conducted a genetic diversity analysis using ISSR markers for some apple genotypes grown in Ardahan region, Turkey. Total genomic DNA (gDNA) isolation from apple leaves was performed using commercial kits. Five ISSR primers were used to determine the genetic diversity among the genotypes studied. Polymerase Chain Reaction (PCR) was performed with all gDNA samples to produce bands to score. PCR products were run in agarose gel and visualized under UV light. Bands on the gels were scored as “1”, while no bands at the corresponding positions were scored as “0”, to generate the matrix file. Five ISSR primers produced a total of 35 bands, and 20 of them were polymorphic. The polymorphic bands rated approximately 57%. Phylogenetic relationships and genetic distances between the genotypes were calculated by using the PAUP [Phylogenetic Analysis Using Parsimony (and Other Methods)] program. According to the PAUP data, the closest genetic distance was 0.03704 between ‘Kaburga’ and ‘Japon Apple’ genotypes, while the furthest genetic distance was 0.48148 between ‘Karanfil Apple’ and ‘Sisli Uruset’. The phylogenetic analysis obtained using UPGMA algorithm produced a phylogenetic tree with two clades. The results suggest that ISSR markers are useful tools for determining genetic relationships among apple genotypes.

Molecular detection of virulent Mannheimia haemolytica and Pasteurella multocida in lung tissues of pneumonic sheep from semiarid tropics, Rajasthan, India

The present study was planned to detect the genetic elements of Mannheimia haemolytica and Pasteurella multocida in pneumonic sheep lungs. Pneumonia was diagnosed on the basis of gross pathological lesions. Lung tissues were collected at necropsy of sheep (n = 96) and subjected to isolation of total DNA. The M. haemolytica -specific PHSSA and Rpt2 genes, and the P. multocidaspecific KMT1 and the Omp87 genes were amplified using polymerase chain reaction (PCR). A housekeeping gene targeting the sheep cellular mitochondrial 12S ribosomal DNA was used as an internal control. PCR reactions were optimized using the positive and negative controls. Gene-specific PCR products were subjected to nucleotide sequencing for confirmation. The pneumonic lungs showed congestion and hemorrhagic changes with consolidation, which was most evident in the whole of the apical lobes and parts of the diaphragmatic lobes. PCR amplification showed detection of PHSSA (327 bp) and Rpt2 (~1022 bp) genes specific to M. haemolytica in 52 (54.1%), and the KMT1 (457 bp) and Omp87 (2627 bp) genes specific to P. multocida in 16 (16.6%) lung samples. Sequence analysis confirmed the PCR products for specific genes. This study highlighted the culture-independent, rapid, and confirmatory diagnosis of ovine pneumonic pasteurellosis caused by M. haemolytica and/or P. multocida .

The GAPDH partial gene of durik-durik (Syzygium sp.) from Riau Province of Indonesia

GAPDH gene encoding glyceraldehyde-3-phosphate dehydrogenase enzyme is one of housekeeping genes which is often used as an internal conntrol in gene expression analysis. This study is aimed to isolate the GAPDH gene from durik-durik (Syzygium sp.). Fresh leaves of Syzygium sp. were collected from Kajuik Lake which is located in Langgam District, Pelalawan Regency, Riau Province, Indonesia. The methods included total DNA isolation using plant DNA isolation kit (Qiagen), polymerase chain reaction, electrophoresis, and sequencing. Data was analyzed using BioEdit7 and MEGA6 softwares, and BLAST program. The DNA partial sequence of GAPDH gene was obtained from Syzygium sp. with the length of 623 bp. The DNA sequence was predicted consisting of 2 exons and 3 introns and encoding 68 amino acids. The DNA sequence of Syzygium sp. GAPDH gene had 74%-89% similartiy to one of other plant species, but none was member of Syzygium genus. The deduced amino acid sequence had 100% similartiy to one of Tropaeolum minus dan Mentha spicata plants. The GAPDH partial gene obtained in this study is the first one reported from Syzygium genus and this sequence can be a basis for isolating of GAPDH gene from other species in Syzygium genus.

Диференціація генотипів сосни звичайної за поліморфізмом довжини інтронів генів дефензинів

Наведено результати дослідження генотипів сосни звичайної (Pinus sylvestris L.) із використанням молекулярних маркерів на підстаі генетичного поліморфізму довжини інтронів генів дефензинів PsDef1-4. Виявлено високу інформативність маркерів на підстаі інтрону гена PsDef2 (IPL-PsDef2) для дослідження внутрішньовидового поліморфізму в сосни звичайної. Встановлено межі осередка інфекції кореневої губки (Heterobasidion annosum (Fr.) Bref.) в сосновому насадженні за допомогою полімеразно-ланцюгової реакції (ПЛР) із використанням видоспецифічних праймерів. Проведено ДНК-профілювання модельних дерев способом електрофоретичного розділення ампліконів IPL-PsDef2 маркерів. За результатами поліморфізму довжини інтронів генів дефензинів здійснено кластеризацію генотипів сосни звичайної із використанням алгоритму UPGMA. на підстаі дендрограми виділено два кластери генотипів зі 100 % бутстреп підтримкою, які відрізняються за стійкістю до кореневої губки. Методом К-середніх визначено значущість кожного з ампліконів для диференціації генотипів сосни на групи. Виявлено амплікони, які можуть бути генетичними маркерами для оцінки потенційної стійкості генотипів сосни звичайної до кореневої губки. Отримані дані щодо поліморфізму локусів генів дефензинів, а також результати кластерного аналізу вказують на перспективність використання IPL-PsDef2 маркерів для генотипування сосни звичайної.

Isolation of Housekeeping Genes on Durik-durik (Syzygium sp)

Housekeeping gene is a gene expressed with a fixed level and in abundant amounts under various conditions. After validation, the housekeeping gene can be used as an internal control to normalize gene expression data. This study reports the isolation of several housekeeping genes in Durik-durik plant (Syzygium sp). This plant material in form of fresh leaves from Durik-durik plants are taken from Kajuik Lake, Riau Province. The next stage is total DNA isolation, polymerase chain reaction, electrophoresis, sequencing and data analysis using bioinformatic tools. The isolated housekeeping genes included 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Beta-tubulin and Ubiquitin with sizes of 400 bp, 679 bp, 1134 bp, 836 bp, 1167 bp and 2155 bp, respectively. In addition to 18S rRNA, the five housekeeping genes are the first reported from the genus Syzygium and referable to isolate housekeeping genes in other species in this genus. The six housekeeping genes can be used as internal controls on Durik-durik plants after validation.

Culture purification and DNA extraction procedures suitable for next-generation sequencing of euglenids

In the present study, five different DNA extraction procedures were examined to determine their effectiveness for extracting DNA suitable for NGS applications. This included two silica-membrane spin column kits, phenol:chloroform, and two CTAB-based methods. Spectrophotometric and fluorimetric measurements as well as standard gel electrophoresis were used as criteria for evaluating the quantity and quality of the isolated DNA prior to the sequencing. Herein, the method of establishing and maintaining axenic Euglena cultures is also presented. The modified CTAB-based method proved to be highly efficient. In terms of DNA quantity and purity (according to the absorbance ratios), the chosen method resulted in DNA of high molecular weight and quality, which fulfills the library construction requirements. Genomic DNA of Euglena hiemalis (CCAP 1224/35) and E. longa (CCAP 1204-17a) isolated using the suggested protocol had been successfully sequenced on the Illumina HiSeq platform. A modified, rapid CTAB-based method of total DNA isolation from Euglena has been described. In terms of the DNA quantity and quality, the protocol devised involving the washing step with DMSO:acetonitrile proved superior to the commonly used, commercially manufactured kits and isolation with phenol:chloroform. The method is also less labor-intensive and time-consuming than the traditional CTAB-based protocol.

No implication of Simian virus 40 in pathogenesis of malignant pleural mesothelioma in Slovenia.

Malignant mesothelioma is predominantly caused by asbestos exposure, although the association of Simian virus 40 in its pathogenesis is currently still under debate. Simian virus 40, a DNA rhesus monkey virus with oncogenic properties, accidentally contaminated early batches of polio vaccine in the 1960s. In the 1990s, viral sequences and proteins were discovered in several human tumors, which triggered research to find a link between Simian virus 40 and human cancers, especially malignant mesothelioma. The aim of our study was to establish an effective laboratory procedure for Simian virus 40 detection and to investigate the presence of Simian virus 40 DNA and small t antigen in mesothelioma samples from Slovenian patients. Paraffin-embedded malignant pleural mesothelioma specimens from 103 Slovenian patients were collected and used for total DNA isolation and real-time polymerase chain reaction for Simian virus 40 small t and large T DNA analysis. Special attention was devoted to primer design, good laboratory practice and polymerase chain reaction contamination prevention. Polymerase chain reaction products were sequenced and BLAST aligned. One 5 microm thick paraffin section from each patient's tissue block was stained with hematoxylin and eosin for histological typing and one for immunohistochemical detection of Simian virus 40 small t antigen using a monoclonal antibody against Simian virus 40 (Pab280). SV40-expressing Wi-38 cells were used as positive control in both PCR and immunohistochemistry. In real-time polymerase chain reaction analyses, only 4 samples gave products with primer pairs amplifying small t antigen and were inconsistent and poorly reproducible. BLAST alignment showed no homology with any deposited SV40 sequences. No immunopositive staining for SV40 small t antigen was found in any of the samples. We found no evidence of SV40 presence in tissue samples from 103 Slovenian patients with malignant pleural mesothelioma. Asbestos exposure remains the main risk factor for malignant pleural mesothelioma in Slovenia.

Діагностика грибів роду Fusarium у сіянцях сосни звичайної методом полімеразної ланцюгової реакції

Найбільш поширеною та небезпечною хворобою сіянців хвойних порід є інфекційне вилягання, збудниками якого є гриби з родів Fusarium, Alternaria, Botrytis та інші. Актуальним завданням є розроблення чутливих і селективних методів ідентифікації патогену. Представлено методику детекції ДНК грибів роду Fusarium у тканинах сіянців сосни звичайної шляхом полімеразно-ланцюгової реакції із видоспецифічними праймерами. Розроблена методика дає змогу визначати 0,1 пг ДНК Fusarium sporotrichella на високому фоні ДНК сосни, що перевищує кількість ДНК патогену в десять тисяч разів. Методику апробовано на різновікових сіянцях сосни звичайної, вирощених у нестерильних умовах закритого ґрунту. Проведено діагностику тканин сіянців сосни, що вилягли, та візуально здорових рослин на присутність ДНК фузаріумів.

ВЫДЕЛЕНИЕ ТОТАЛЬНОЙ ДНК БАКТЕРИАЛЬНЫХ СООБЩЕСТВ ДЛЯ ФОРМИРОВАНИЯ КОЛЛЕКЦИИ ХЕМОЛИТОТРОФНЫХ МИКРООРГАНИЗМОВ МЕСТОРОЖДЕНИЯ ШАНУЧ (КАМЧАТКА)

ВЫДЕЛЕНИЕ ТОТАЛЬНОЙ ДНК БАКТЕРИАЛЬНЫХ СООБЩЕСТВ ДЛЯ ФОРМИРОВАНИЯ КОЛЛЕКЦИИ ХЕМОЛИТОТРОФНЫХ МИКРООРГАНИЗМОВ МЕСТОРОЖДЕНИЯ ШАНУЧ (КАМЧАТКА)

Bacterial community in ancient permafrost alluvium at the Mammoth Mountain (Eastern Siberia)

Permanently frozen (approx. 3.5Ma) alluvial Neogene sediments exposed in the Aldan river valley at the Mammoth Mountain (Eastern Siberia) are unique, ancient, and poorly studied permafrost environments. So far, the structure of the indigenous bacterial community has remained unknown. Use of 16S metagenomic analysis with total DNA isolation using DNA Spin Kit for Soil (MO-Bio) and QIAamp DNA Stool Mini Kit (Qiagen) has revealed the major and minor bacterial lineages in the permafrost alluvium sediments. In sum, 61 Operational Taxonomic Units (OTUs) with 31,239 reads (Qiagen kit) and 15,404 reads (Mo-Bio kit) could be assigned to the known taxa. Only three phyla, Bacteroidetes, Proteobacteria and Firmicutes, comprised >5% of the OTUs abundance and accounted for 99% of the total reads. OTUs pertaining to the top families (Chitinophagaceae, Caulobacteraceae, Sphingomonadaceae, Bradyrhizobiaceae, Halomonadaceae) held >90% of reads. The abundance of Actinobacteria was less (0.7%), whereas members of other phyla (Deinococcus-Thermus, Cyanobacteria/Chloroplast, Fusobacteria, and Acidobacteria) constituted a minor fraction of reads. The bacterial community in the studied ancient alluvium differs from other permafrost sediments, mainly by predominance of Bacteroidetes (>52%). The diversity of this preserved bacterial community has the potential to cause effects unknown if prompted to thaw and spread with changing climate. Therefore, this study elicits further reason to study how reintroduction of these ancient bacteria could affect the surrounding ecosystem, including current bacterial species.

Application of rps16 Intron and trnL-trnF Intergenic Spacer Sequences to Identify Rengas clone Riau

<p>Rengas clone Riau has been identified using morphological characters and molecular technique with a <em>psbA-trnH</em> intergenic spacer, however, this method can only determine its taxonomic status at genus level, namely <em>Gluta</em> sp. This study reports application two DNA barcodes, i.e. <em>rps16 </em>intron<em> </em>and <em>trnL-trnF</em> intergenic spacer, to identify Rengas clone Riau. The methods included collection of the leaves from Kajuik Lake, total DNA isolation, electrophoresis, PCR (polymerase chain reaction), gel purification and sequencing. The <em>rps16 </em>intron size was 659 bp and the <em>trnL-trnF </em>intergenic spacer was 527 bp. The BLASTn analysis showed that sequences of the <em>rps16 </em>intron and the <em>trnL-trnF </em>intergenic spacer of <em>Gluta</em> sp clone Riau had 100% similarity to those of <em>G. renghas </em>deposited in GenBank. These results were supported by high max score, high total score, query cover = 100%, and E-value = 0. The dendrograms also showed the closest relationship of <em>Gluta</em> sp clone Riau with <em>G. renghas </em>deposited in GenBank compared to other species of <em>Gluta</em>. In conclusion, this study succeeded in identifying Rengas clone Riau as <em>Gluta renghas</em> by using sequences of the <em>rps16 </em>intron and the <em>trnL-trnF</em> intergenic spacer. A combination of DNA barcodes could be applied to identify various plants as long as the database for the DNA barcodes is available in public database such as GenBank. </p>

Detection of exogenous gene doping of IGF‐I by a real‐time quantitative PCR assay

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study.

2. Characterization Of Lactic Acid Bacteria (LAB) Origin Sumatran Orangutan (Pongo abelii) In Zoo Bukittinggi Based On Analysis 16 S rRNA

The purpose of this study was to characterize the bacterial 16S rRNA gene of Sumatran orangutan (Pongo abelii) Bukittinggi West Sumatera zoo. The sample used in this study are lactic acid bacteria of the Sumatran orangutan (Pongo abelii) derived from zoo Bukittinggi West Sumatra Indonesia. This study was an exploratory study that conducted at the Laboratory through several stages. The first stage was the isolation of LAB from faeces of Sumatran orangutans using MRS agar medium and then cultured in a liquid medium NB. The next stage was the isolation of total DNA, and then, the third stage was the amplification of the 16S rRNA gene and agarose gel electrophoresis. Then,in the fourth stage of determining the DNA sequence and analysis of DNA sequence homology. The final stage was the computational analysis of 16S rRNA gene Sumatran orangutan (Pongo abelii). The results showed that lactic acid bacteria (LAB) from the Sumatran orangutan (Pongo abelii) is close to lactic acid bacteria Lactobacillus helveticus strain IMAU50151 with the levelof similarity of 89%. It is possible that these bacteria is a new species or the species that LAB has not been reported in Genbank.