Genomic DNA Purification Research Articles

Comparison of DNA extraction methods from Geranium (Geraniaceae)

The genus Geranium (Geraniaceae); with about 320 species throughout the temperate regions, is chemically characterised by the presence of tannins, flavonoids, anthocyanins and essential oils which interfere with the extraction of pure genomic DNA. It is necessary to optimise the extraction protocols to reduce the effects of the presence of these compounds to the lowest level. The present study compares the plant genomic DNA extraction Kit (DNP™ Kit), CTAB DNA extraction method by Murray and Thompson and Sahu et al., from the extracting DNA point of view Geranium species. The results showed significant differences in DNA contents between the three methods. Quantity and quality of extracted genomic DNAs were compared by employing the spectrophotometer, Nano-Drop, agarose gel electrophoresis, and polymerase chain reaction (PCR) methods and molecular marker such as (ITS and trnL-F) and ISSR. The method of Sahu et al., provided the best results (200 ng/µL) in terms of quantity and quality of DNA, therefore, this method was taken and optimised for DNA extraction. Our results proposed that this method could be effective for plants with same polysaccharides, proteins and polyphenols components. The advantage of this method is that it omits the use of liquid nitrogen and toxic phenols which are expensive. The success of this method in obtaining high-quality genomic DNA has been demonstrated in the Geranium species group and the reliability of this method has been discussed.

Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9.

The recovery of non-functional-enhanced green fluorescence protein can be used as indicator to facilitate the identification of mutants generated by CRISPR/Cas9. The CRISPR/Cas9 system is a powerful tool for genome editing and it has been employed to knock out genes of interest in multiple plant species. Identification of desired mutants from regenerated plants is necessary prior to functional study. Current screening methods work based on the purification of genomic DNA and it would be laborious and time consuming using these methods to screen mutants from a large population of seedlings. Here, we developed the non-functional enhanced green fluorescence protein (nEGFP) reporter gene by inserting a single guide RNA (sgRNA) and the protospacer adjacent motif in the 5' coding region of EGFP, and the activity of nEGFP could be recovered after successful targeted editing. Using the nEGFP as the reporter gene in Nicotiana tabacum, we found that over 94% of the plants exhibiting EGFP fluorescence were confirmed to be desired mutants. The use of this nEGFP reporter construct had limited negative effect on editing efficiency, and the expression of Cas9 and sgRNA was not affected. Moreover, this method was also applied in grape by targeting the phytoene desaturase gene(PDS), and the grape cells with EGFP signal were revealed to contain targeted mutations in VvPDS. Our results show that the nEGFP gene can be used as reporter to help screen mutants according to the recovered EGFP fluorescence during the application of CRISPR/Cas9 in plants.

Development of a loop-mediated isothermal amplification assay targeting lmo0753 gene for detection of Listeria monocytogenes in wastewater.

Contaminated wastewater plays an important role in the transmission of Listeria monocytogenes in the environment. In this study, a loop-mediated isothermal amplification (LAMP) assay for sensitive detection of L.monocytogenes in wastewater from treatment plants was developed, validated and compared to conventional PCR. The lmo0753 gene which codes for a Crp/Fnr family transcription factor, was targeted to design four specific primers to detect L.monocytogenes in 60min at 63°C in a water bath. Amplification products were visualized by agarose gel electrophoresis. The detection limit of the LAMP assay was 65fgµl-1 of DNA and 38CFU per ml. Conventional PCR was 10 times less sensitive than LAMP assay with primers targeting the HlyA gene. A total of 70 crude wastewater samples collected at different treatment stages (aeration tank, pre chlorination and post chlorination), were tested directly by LAMP and PCR. Samples from aeration and pre-chlorination stages tested positive with LAMP and culture method but not with conventional PCR. LAMP assay was tolerant to inhibitors present in wastewater and circumvented the need for isolation of pure DNA for detection. Both LAMP assay and culture method failed to detect L.monocytogenes in post-chlorinated wastewater, confirming the efficiency of the treatment process in the removal of L.monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated wastewater effluent contains Listeria monocytogenes which survives conventional wastewater treatment processes and can re-enter human food chain, thus it is imperative to detect L.monocytogenes using a rapid and an inexpensive method. To the best of our knowledge, this is the first report of a loop-mediated isothermal amplification (LAMP) assay, targeting the lmo0753 gene for detection of L.monocytogenes in wastewater from treatment plants. The LAMP assay detects L.monocytogenes in 60min at 63°C in a water bath. LAMP does not require isolation of pure genomic DNA hence it is a user friendly method for L.monocytogenes detection.

Keragaman genetik puyuh Jepang (Coturnix japonica) berdasarkan analisis sekuen DNA mitokondria gen Cytochrome-b

Coturnix japonica is a Japanese quail which is commonly raised in Indonesia. The aim of the study was to determine the genetic diversity of Japanese quail ( Coturnix japonica ) based on complete sequence of mitochondrial DNA Cytochrome b gene analysis. This study used 12 samples of quail blood collected from two different village breeding centres (VBC) representing black and brown plumage lines. Total DNA genome was isolated from whole blood by following the Wizard® Genomic DNA Purification Kit protocol (Promega, USA). The DNA amplification process in this study was carried out using novel forward and reverse primers using Primer3 software. Additionally, PCR products were sequenced and analysed using MEGA 6.0 and DNAsp v.5 softwares. A total of 21 sequences consists of 12 sequences of quail sample and 9 reference sequences obtained from NCBI website were analyzed to construct phylogenetic tree and to evaluate genetic diversity of Japanese quails. The sequence analysis showed that Japanese quail population in this study had kinship with Francolinus pintadeanus and Coturnix chinensis with an 81% bootstrap value. The genetic distance among Japanese quails was ranged from 0.000 to 0.003. In addition, the mean of genetic distance for Japanese quail was 0.002 and the mean of genetic distance for 21 samples was 0.067. Tajima D'test values were -1.35536 for Japanese quail population and -1.38090 for whole population used in this study. The conclusion of this study was the Japanese quail had low genetic diversity based on complete sequence of Mitochondrial DNA Cytochrome b gene analysis that mean Japanese quail had low level of evolution

<p class="Body">Comparison of five DNA extracting protocols from Typhlodromus (Anthoseius) bagdasarjani Wainsten and Arutunjan (Acari: Phytoseiidae)

Obtaining clean DNA from mites to be used in molecular studies has always been a challenging task. In this study, five protocols (CTAB-based handmade kit, salting out protocol, STE protocol, modified CTAB-based protocol and wizard Genomic DNA purification kit) were evaluated to extract DNA from Typhlodromus (Anthoseius) bagdasarjani. Quantitative and qualitative analyses of the DNA concentrations and a comparison between the ratio of absorption of wavelength from 260 to 280 nm in a completely randomized split plot design for five extraction protocols and three levels of 1, 5, 10 mites were performed. PCR protocol was used to examine the amplification of the ITS1 fragment from the specimens. The results showed that wizard kit and CTAB modified protocol were the bests. However, economically, modified CTAB-based protocol is the best choice for extensive experiments, since this protocol allows extracting DNA from an individual mite and provides the highest concentration DNA (143.35±0.85ng) and the best OD (1.80±0.03) for molecular studies.

Comparative account of DNA extraction protocols in some freshwater prawns of Genus Macrobrachium (Bate, 1868) (Family Palaemonidae) from Jammu waters for PCR based applications

Genetic variations among prawns act as an important tool to characterize and differentiate between the species. Molecular and phylogenetic analysis of shrimps and prawns like any other organism rely on high yields of pure and better quality genomic DNA. In this regard isolation of DNA is the first and basic step. In spite of the availability of many protocols of DNA extraction from animal tissues, it is difficult to ascertain that which one would provide desired results for prawn tissue. In the present study, three different techniques of DNA isolation i.e., salting out, phenol-chloroform and Qiagen DNA extraction kit were performed and compared for their yield. Cephalothoracic tissue and muscle tissue of pleopods were used for isolation. Tissue samples from fresh specimens as well as from alcohol preserved specimens were employed for extraction. The quantity (µg/ml) and quality of isolated DNA were determined by UV spectrophotometry and agarose gel electrophoresis. Results showed that Phenol-chloroform method with slight modifications obtained higher yield of genomic DNA as compared to other methods. The present work also revealed that among fresh specimens cephalotoracic tissue yielded high concentration DNA than muscle tissue. However, among alcohol preserved specimens, the concentration of DNA was higher in muscle tissue of pleopods. The high quality DNA was then subjected to randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) analysis. The DNAs produced clear, sharp and reproducible PCR (Polymerse chain reaction) product pattern.

Comparative Analysis of Different DNA Extraction Methods and Preliminary Analysis of Genetic Diversity of Hedera nepalensis K. Koch. in Vietnam Based on GBSSI Marker

Though the ivy (Hedera nepalensis K. Koch.) has long been utilized in traditional medicine, its genome information is very limited. For plants, an effective method of DNA extraction is a very important step which greatly affects subsequent genetic analyses. In this study, four different methods of DNA extraction from dry leaves were used. A comparison of different protocols resulted in the yield of extracted DNA that ranged from 10.5 to 437.4 ng/μl and with a purity ranged from 1.8 to 2.2. Based on the PCR results of GBSSI gene, Gene JET Plant Genomic DNA Purification Mini Kit is the most optimal extraction method for Vietnam ivy’s dry leaves. A preliminary analysis of the phylogenetic tree based on the GBSSI marker showed that ivy growing in a number of northern mountainous provinces of Vietnam belonged to the H. nepalensis K. Koch species. The high - quality total DNA will allow us to amplify different DNA markers, providing valuable genetic information to preserve and develop medicinal resources in Vietnam.
 Keywords
 GBSSI, Hedera nepalensis K. Koch, DNA extraction.
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Establishment of a Rapid Detection Method for Rice Blast Fungus Based on One-Step Loop-Mediated Isothermal Amplification (LAMP)

Rice blast is one of the most serious diseases for rice, and controlling the filamentous fungus Magnaporthe oryzae that causes rice blast is crucial for global food security. Typically, early infected rice does not show symptoms. Therefore, the early diagnosis of rice blast is particularly important to avoid uncontrollable propagation of rice blast fungus. In the present work, a rapid and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect the pathogen at the early infected stage of rice. The Alb1 superfamily hypothetical protein MGG_04322, a nuclear shuttling factor involved in ribosome and melanin biogenesis, was chosen as the target for designing the LAMP primers. The LAMP assay enabled rapid detection of as little as 10 pg of pure genomic DNA of M. oryzae. In addition, we established the quantitative LAMP (q-LAMP) detection system to quantify the conidia of rice blast fungus. The q-LAMP assay enabled rapid detection (within 35 min) of the fungal spores at a sensitivity of 3.2 spores/ml. In addition, the assay sets up the linearization formula of the standard curve as y = 0.3066 + 15.33x (where x = amplification of time), inferring that spore number = 100.60y. In addition, the q-LAMP assay was successfully used to detect the presence of the virulence strains of M. oryzae (wild type) in comparison with that of the two mutant strains by quantifying the biomass within host tissue. These results provide a useful and convenient tool for detecting M. oryzae that could be applied in the incubation period of rice blast before symptoms appear.

PF501 HOMOZYGOUS I281T MUTATION IN HJV CAUSING INDOLENT JUVENILE HEMOCHROMATOSIS WITH A MIDDLE TO LATE ONSET

Background:Juvenile hemochromatosis (JH; hemochromatosis type 2A) is a rare autosomal recessive disorder of systemic iron overload due to homozygous mutations of HJV (HFE2) which encodes hemojuvelin, an essential regulator of hepcidin expression, leading to intestinal iron hyperabsorption because of hepcidin deficiency. JH usually occurs younger than 30 years old, and causes liver fibrosis, hepatocellular carcinoma, diabetes, hypogonadism and heart failure with fatal outcomes.Aims:Here we report two Japanese siblings with JH caused by a homozygous I281T mutation in HJV, who unusually showed an indolent clinical course with a middle to late onset.Methods:After obtaining a written informed consent from each individual, blood samples were collected, followed by genomic DNA purification, and the coding regions of HFE, HFE2 (HJV), HAMP, TFR2, and SLC40A1 were sequenced. Genetic mutation was then evaluated by comparing the sequencing results to open access genetic information on the NCBI websites.Results:Patient #1 is a 38‐year‐old woman, who was referred to us due to 1‐year history of amenorrhea, thirst and weight loss. The patient's laboratory findings showed high levels of transferrin saturation (93%) and serum ferritin (2274 ng/mL), insulin‐dependent diabetes, iron deposition of the anterior pituitary gland by nuclear magnetic resonance (NMR) with hypogonadism, and massive iron deposition in hepatocytes by liver biopsy. Phlebotomy effectively improved iron overload as a treatment of hemochromatosis and has been continued for more than 15 years on an outpatient basis. Patient #2, aged 55, is an elder sister of the patient #1, who referred to us because due to liver dysfunction, dyslipidemia and hyperglycemia for 3 years and thirst for 1 year. She was also found to have high levels of transferrin saturation (92%) and serum ferritin (4340 ng/mL), insulin‐dependent diabetes, iron deposition of the pituitary gland by NMR with hypogonadism, massive iron deposition in hepatocytes by liver biopsy, and cardiomyopathy with congestive heart failure. Phlebotomy successfully improved organ damages associated with iron overload and has been continued for more than 4 years on an outpatient basis. It was later found that serum levels of hepcidin were below the measurement sensitivity in both patients.To identify the causative genetic defect responsible for the iron overload in these two siblings (probands), we screened them for mutations in the genes involved in hereditary hemochromatosis as well as their healthy brother, and two healthy sons of the patient #1. Both probands were homozygous for a missense mutation in HJV exon 4 (842 T>C; I281T). Both of the patient #1's sons were heterozygous for I281T of HJV, and the brother had no mutations related with hemochromatosis.Summary/Conclusion:We have reported two Japanese siblings (onset, 37 and 52 years) with the homozygous mutation of HJV I281T in middle to late onset with an indolent clinical course. HJV mutations, which are the cause of JH, have been widely identified mainly in a Caucasian population. However, the homozygous mutation of HJV I281T as observed in the current study has only been identified in one Greek patient (Papanikolaou G. Nature genet. 2004), whose JH notably occurred at the age of 39 years and was diagnosed at the age of 49 years. Therefore, together with the current findings, it was suggested that the homozygous mutation of I281T may be associated with an indolent clinical course and a middle to late onset of JH.

All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes.

Meloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both transcriptomics and cellular biology. Among others, it was confirmed that a generalized gene repression is a hallmark of early developing GCs formed by Meloidogyne javanica in Arabidopsis and tomato. One of the main mechanisms behind this gene repression involve small RNAs (sRNAs) directed gene silencing. This is supported not only by the described action of several microRNAs differentially expressed in galls, but by the differential abundance of 24-nucleotide sRNAs in early developing Arabidopsis galls, particularly those rasiRNAs which are mostly involved in RNA-directed DNA methylation. Their accumulation strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls. However, the contribution of this global gene repression to GCs/galls formation and maintenance is still not fully understood. Further detailed studies, as the correlation between gene expression profiles and the methylation state of the chromatin in galls are essential to raise testable working hypotheses. A high quality of isolated DNA and RNA is a requirement to obtain non-biased and comprehensive results. Frequently, the isolation of DNA and RNA is performed from different samples of the same type of biological material. However, subtle differences on epigenetic processes are frequent even among independent biological replicates of the same tissue and may not correlate to those changes on the mRNA population obtained from different biological replicates. Herein, we describe a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample adapted to our biological system. The quality of both nucleic acids from Arabidopsis galls formed by M. javanica was high and adequate to construct RNA and DNA libraries for high throughput sequencing used for transcriptomic and epigenetic studies, such as the analysis of the methylation state of the genomic DNA in galls (MethylC-seq) and RNA sequencing (RNAseq). The protocol presents guidance on the described procedure, key notes and troubleshooting.

Detection of Golovinomyces orontii using species-specific primers and high-resolution melting analysis

This study presents the development of species-specific PCR primers for detection of Golovinomyces orontii, the causal agent of powdery mildew disease on different cucurbit hosts. The species-specific primers CgF2 and CgR2 were designed based on partial ITS and 5.8S rDNA sequences using an isolates collection of powdery mildew and downy mildew pathogens from different sources. After optimization of the PCR conditions, a 233 bp fragment was amplified only from DNA of G. orontii. Limit of detection was determined to be 0.01 ng of pure conidial genomic DNA. Further association of PCR and high-resolution melting (HRM) analysis showed that the designed primer set can be used for differentiation of G. orontii from different powdery mildew fungi in infected cucurbits tissue collected in the field.

Mutational analysis of key genes in patients from the Kazakh population with early onset of breast cancer.

e13038 Background: Breast cancer (BC) represents the most common malignancy and has the highest mortality among women, both in the world and in Kazakhstan. Approximately 20-30% of cases of hereditary BC are caused by presence of BRCA1/2 genes defects. Also, there are additional genes which can increase the risk of BC and they are still under study.The aim of this study was to identify new, detectable and objective markers of key cancer genes by next-generation sequencing (NGS) of young BC patients. Methods: Blood was collected from 129 BC patients (average age 34.87±5.587). DNAs were extracted using GeneJET Genomic DNA Purification Kit. NGS was done on the MiSeq platform using TruSightCancer Kit. NGS data were analyzed by the MiSeq Reporter v. 2.4 software. Variant annotation and interpretation was done by Variant Studio 3.0 software. Results: We perform the NGS and did the detailed bioinformatics analysis of sequences of 16 key genes of BC (BRCA1/BRCA2, BRIP1, ATM, CDH1, CHEK2, NBN, NF1, PALB2, STK11, TP53, RAD51C, RAD51D, BARD1, MUTYH and FANCC). At all 3016 variants were detected, 1080 (35.8%) out of which were missense; 1494 (49.5%) synonymous; 17 (0.6%) frameshifts; 2 (0.06%) inframe deletions, 8 (0.3%) stop gained; 4 (0.1%) splice variant, 411 (13.6%) intron or non-coding variants. We identified 24 pathogenic and likely pathogenic mutations in 31 patients (24% of all patient cohort), 3 mutations are novel. All mutations were in heterozygous state and were rare or had frequency data not available (NA) in 1000G, ESP6500and ExAC databases. Analysis of mutation type revealed 17 frameshift mutations, 2 missense mutations, 8 stop gained mutations, 4 mutations involving uncorrected splicing. Out of the 31 pathogenic and likely pathogenic variants, were detected 14 in BRCA1, 9 in BRCA2, 4 in TP53 genes, 3 in PALB2 and 1 in NBN. (BRCA2: c.6468_6469delTC, c.7567_7568delCT, c.9976A > T, c.8174G > A, c.2442delC, c.2808_2811delACAA, c.9253delA, c.9409dupA and c.6058G > T; BRCA1: c.2498delT, c.5224C > T, c.3020C > G, c.3352C > T, c.5530+1G > A, c.5341-2delA and BRCA1-c.5329dupC in 6 cases; NBN: c.657_661delACAAA; PALB2: c.172_175delTTGT, c.1034T > G and c.18_22delGAAGC; TP53- c.156dupA, c.1024C > T, c.844C > T and c.584T > C). The most frequent genes were BRCA1 (45%) and BRCA2 (29%). Conclusions: The study focused on identifying mutation profile of early onset BC using NGS. Clinically significant variants was detected which are promising biomarkers for the successful diagnosis and therapy of BC at women in reproductive age.

Preparation and purification of DNA from bacterial cells; characterization of plasmid DNA

The use of genetic material to deliver genes for therapeutic purposes has been practiced for many years. With the advancement in genetic engineering, foreign genes of industrial applications can be inserted into cloning vector for mass production in various host cells. Escherichia coli is an extremely important model organism in modern biological engineering, the suitable growth media is essential for the optimal expression of the genes in E. coli . The present study aims at isolation and purification of genomic DNA from E. coli , the characterization of pBR322 plasmid DNA. Bacterial culture conditions were optimized in shake – flask cultures based on optimal temperature, inoculum size and medium composition. Solutions and methods are disclosed for the effective, simple isolation of DNA from bacterial cells. High bioprocess recovery and product quality were primarily associated with the complete removal of total cellular RNA impurity. The process was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and other impurities were removed by selective precipitation using calcium chloride at an optimal concentration. The optimal conditions for the growth of Escherichia coli were shown maximum absorbance as 7.5 at 37 0 C temperature, 1% inoculum size using TB medium composition. The purified genomic DNA had concentration as 73.5 µg/ml and purity 1.8. The 0.5M CaCl 2 was optimal concentration for removal of RNA. The plasmid DNA pBR322 was confirmed by comparing the band to 4.36 Kb, purity of plasmid was 1.85 and it contains 96.8% of super coiled DNA. The contaminants like chromosomal DNA, RNA, host cell proteins and mycoplasma were absent in the plasmid DNA.

A Duplex PCR Assay for Rapid Detection of Phytophthora nicotianae and Thielaviopsis basicola.

A duplex PCR method was developed for simultaneous detection and identification of tobacco root rot pathogens Phytophthora nicotianae and Thielaviopsis basicola. The specific primers for P. nicotianae were developed based on its internal transcribed spacer (ITS) regions of ribosomal gene, ras gene and hgd gene, while the specific primers for T. basicola were designed based on its ITS regions and β-tubulin gene. The specificity of the primers was determined using isolates of P. nicotianae, T. basicola and control samples. The results showed that the target pathogens could be detected from diseased tobacco plants by a combination of the specific primers. The sensitivity limitation was 100 fg/μl of pure genomic DNA of the pathogens. This new assay can be applied to screen out target pathogens rapidly and reliably in one PCR and will be an important tool for the identification and precise early prediction of these two destructive diseases of tobacco.

Genetic diversity of Ongole Grade, Aceh, and Sumbawa cattle based on polymorphism on ND-5 fragment mitochondrial DNA using PCR-RFLP technique

Abstract. Sutarno, Zahrah S, Astirin OP, Herawati E, Setyawan AD. 2019. Genetic diversity of Ongole Grade, Aceh, and Sumbawa cattle based on polymorphism on ND-5 fragment mitochondrial DNA using PCR-RFLP technique. Biodiversitas 20: 783-788. Genetic diversity is the basis of livestock breeding because it can be used as an initial improvement in livestock quality through artificial selection. This study aims to determine polymorphism in ND-5 fragment of mitochondrial DNA in Ongole Grade, Aceh, and Sumbawa cattle and their genetic diversity. The total DNA from the blood of the local cattle was extracted using the Wizard genomic DNA purification system from Promega and amplified using the PCR technique. The PCR product was then digested with HindIII enzyme using the RFLP technique to detect polymorphism. The genetic diversity of the Ongole Grade, Aceh, and Sumbawa cattle was analyzed using the formula from Nei and its genetic relationship was evaluated with the 2.02i NTSYSpc version program. Our findings showed there were polymorphisms in the ND-5 fragment of mitochondrial DNA. Digestion with HindIII restriction enzyme produces two types of haplotypes. Haplotype B is a 453 bp-sized DNA fragment that is not truncated by the HindIII enzyme, and haplotype A is a DNA fragment cut by HindIII enzyme into two with fragments of 336 bp and 117 bp. Polymorphism was found in Ongole Grade cattle, but not in Sumbawa and Aceh cattle. Haplotypes diversity in ND-5 fragments of mitochondrial DNA of Ongole Grade was 0.6250 while Sumbawa and Aceh cattle displayed no diversity of haplotypes. The genetic relationship shows that Sumbawa cattle belonged to the same cluster with Ongole Grade but separated from Aceh cattle.

Identificación de marcadores microsatelites polimórficos en el genoma de Dioscorea trífida L.f. “sachapapa”

Identificación de marcadores microsatelites polimórficos en el genoma de Dioscorea trífida L.f. “sachapapa” Identification of marking polimorfic microsatellites in the genome of Dioscorea trífida Lf. “sachapapa” Roberson Ramírez, Pedro Adrianzén, Marianela Cobos y Juan Castro Centro de Investigaciones de Recursos Naturales (CIRNA) Universidad Nacional de la Amazonia Peruana (UNAP), Apartado postal 496S Facultad de C. Biológicas DOI: https://doi.org/10.33017/RevECIPeru2011.0049/ RESUMEN El objetivo principal del estudio fue identificar y evaluar marcadores microsatélites polimórficos presentes en el ADN genómico de D. trífida L.f. “sachapapa”. Para esto se modificó el protocolo CTAB 2X que permitió obtener ADN genómico de buena calidad con un ratio de 1,78 y una cantidad de 186,9 g/ml, mostrando ser aceptable para la amplificación de los microsatelites; del mismo modo se estandarizaron y amplificaron los marcadores microsatélites polimórficos, con seis pares de iniciadores, cuyos productos de amplificación variaron en un rango de 100 a 200 pb. Los 6 pares de iniciadores microsatélites utilizados no mostraron productos inespecíficos, resultando los iniciadores MTI3 y MTI10 más polimórficos con un contenido de información polimórfica (PIC) de 0,84 y 0,81 respectivamente. Sin embargo; el iniciador MTI4 resultó con menor polimorfismo con un PIC de 0,52. Se encontró una ligera dependencia entre el número de alelos diferentes y el contenido de información polimórfica (PIC) con un índice de correlación relativamente alta (r 2= 0,69). Se concluye que la técnica de obtención y purificación de ADN genómico de D. trífida L f. es ideal para extraer ADN genómico, asimismo los 6 pares de marcadores microsatélites estandarizados son adecuados para futuros trabajos a nivel molecular en esta especie. Descriptores: Microsatélites, Dioscorea trífida L f., ADN genómico, PIC. ABSTRACT The main objective of the study was to identify and evaluate polymorphic microsatellite markers present in genomic DNA D. trífida Lf. To modify this was achieved 2X CTAB protocol that yielded genomic DNA good quality with a ratio of 1.78 showing to be acceptable for the amplification and an amount of 186.9 g/ml of the microsatellites, the same way were standardized and polymorphic microsatellite markers amplified with six pairs of primers, whose amplification products varied in the range of 100 to 200 bp. The 6 microsatellites primer pairs showed no nonspecific products, resulting initiators MTI10 MTI3 and more polymorphic polymorphic information content (PIC) of 0.84 and 0.81 respectively. However; the initiator was less polymorphism MTI4 with a PIC of 0.52. There was a slight dependence between the number of different alleles and polymorphic information content (PIC) with a relatively high correlation index (r2 = 0.69). We conclude that the technique of production and purification of genomic DNA from D. trífida L f. is ideal for extracting genomic DNA, also the 6 pairs of microsatellite markers are adequate standard for future work at the molecular level in this species. Keywords: Dioscorea trífida L f., Microsatellite, genomic DNA, PIC.

Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation.

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

The Antibacterial Effect of Silver and Zinc Oxide Nanoparticles against Intracellular Brucella melitensis

Background: Brucellosis is a zoonotic disease of worldwide importance among animal and humans. The most important virulence factor of Brucella melitensis is related to intra-macrophage survival. On the other hand, the side effects of the current brucellosis treatment regime, it is necessary to find out new antimicrobial agents to treat the disease.Objective: To Isolate Brucella melitensis from local white cheese and raw milk by culture method with identification using PCR and determination of their susceptibility to classical antibiotics and silver and zinc oxide nanoparticles.Materials and methods: A total of 150 local soft cheese and raw milk samples collected from different local markets in Erbil city and were examined for the presence of Brucella melitensis during a period of (6) months (November 2015 to April 2016).Selective media; such as, Brucella agar were used for the isolation, and identified according to standard biochemical tests and confirmed by PCR.Antibiotic susceptibility test determined by Kirby-Bauer disk diffusion method and the antimicrobial activity of silver and zinc oxide nanoparticles were carried out in Muller–Hinton agar with 1% sheep’s blood by well diffusion method. Five mm diameter wells were prepared and loaded with Ag(20 nm size) and ZnO (20 nm size) nanoparticles dilutions, finally the combination of ampicillin/ cloxacillin with the nanoparticles were tested against isolates of Brucella melitensis.Results: Out of the 150 food samples, 53(35.33%) were contaminated; 21(28%) of the total samples of local soft cheese and 32(42.6%) of raw milk was contaminated with Brucella melitensis. Twenty isolates were identified according to standard biochemical tests, and confirmed by PCR for detection of pure Brucella genomic DNA in PCR. Brucella-specific primer BgF/BgR, were evaluated for detection of pure Brucella genomic DNA, provided bands on agarose gel corresponding to a 208 base pair product when compared to molecular ladder, the specific primer IS711were detected for Brucella melitensis, and achieved by given bands on agarose electrophoresis corresponding to 731 base pair from the total isolates. Seventeen from 20 isolates were found positive for Brucella with BgR/BgF primers and 15 isolates were positive for IS711 primer. The sensitivity to antibioticsshowed that the resistance to ampicillin and ceftazidime were (85%), for aampicillin/cloxacillin was (45%), and for imipenem and piperacillin were 80% and 90% respectively. The inhibition zone of ZnO-NP against Brucella melitenisis ranged between 10-20 mm when the concentration was 10-1000 ppm. While the inhibition zone of Ag NP was 11.5-24.5 mm. Combination of amoxicillin/ cloxacillin with nanoparticles show a synergistic effect as demonstrated by the increase of inhibition zone to 20 mm fir ZnO-NP and 23 mm for AgNP nanoparticles.Conclusion: Brucella melitensis contaminate local white soft cheese and raw milk and showed variable sensitivity to antibiotics and some isolates were resistant to ampicillin/ cloxacillin, ceftazidime, imipenem, piperacillin, and trimethoprim-sulphamethoxazole. The present study data show that silver and zinc oxide nanoparticles inhibit the growth of Brucella melitensis. The combination of silver nanoparticles and ampicillin/ cloxacillin showed a synergistic effect.

So sánh và đánh giá các phương pháp ly trích DNA trong các sản phẩm có nguồn gốc từ hạt ca cao

Four methods for extracting DNA in cocoa-derived products (cocoa powder, cocoa butter, cocoa mass, and dark chocolate) are described. These DNA extraction methods included four commercial kits DNeasy Plant kit, DNeasy Plant kit with polyvinyl polypyrrolidone (PVPP), QIAamp DNA Stool kit, Wizard Genomic DNA Purification kit. The DNA yield, purity, and quality for four different cocoa matrices are discussed. The yield and purity were determined through spectrophotometry: DNA concentration, DNA purity and PCR positive amplification. Three different conventional PCR reactions were used to assess the quality of the DNA extracted. The Wizard Genomic DNA Purification kit was found to be the best for DNA quality and PCR detection in cocoa powder and dark chocolate ([DNA] = 44.83 (ng/µL); A260/A280 = 1.4-1.7, PCR positive amplification) and ([DNA] = 29.34 (ng/µL) and A260/A280 = 1.4-1.7, PCR positive result, respectively). The cocoa butter matrix gave the lowest DNA yield, and negative amplification results for three the examined extraction methods DNeasy Plant kit with PVPP, DNeasy Plant kit without PVPP, and QIAamp DNA Stool kit [DNA]

Genetic identity based on simple sequence repeat (SSR) markers for Quinoa (Chenopodium quinoa Willd.)

Molecular markers based on simple sequence repeats (SSRs) constitute a highly effective instrument in the identification of quinoa genotypes ( Chenopodium quinoa ), and they are very useful in the management and conservation of germplasm banks. The present study was carried out in the Molecular Biology Laboratories of the Megalaboratory of the National University of the Altiplano and the National Agrarian University la Molina. With the objective of determining a minimum group of highly informative initiators for the cultivation of quinoa to study and identify the obtained alleles and implementing and incorporating this technology into research of genetic identity, the molecular analysis of nine loci located by microsatellite markers (SSRs) was performed on a sample of 26 varieties of quinoa: Ayrampo, Amarilla de Marangani, Choclito, Chullpi, Huariponcho, Pandela, Sajama, Witulla, Kcancolla, Negra Collana, Salcedo, Pasankalla, Blanca de Juli, and Chenopodium petiolare from the CIP-Camacani and Blanca de Juli, Kcancolla, Negra Collana, Pasankalla, Altiplano, Illpa INIA, Salcedo, Ayara Blanca de Juli, Ayara Blanca de Arequipa, Ayara Cancolla, Ayara Pasankalla, and Ayara Salcedo from the INIA. Genomic DNA was extracted by PCR (GeneJET Plant Genomic DNA Purification), and 20 microsatellite regions were amplified. The amplified fragments were loaded on polyacrylamide gels to determine their size in base pairs, of which only nine showed products with reading quality (QCA012, QCA015, QCA021, QCA029, QCA034, QCA040, QCA053, QCA055 and QCA067). The fragments were evaluated for their allelic richness, heterozygosity (H) and polymorphic information content (PIC). The data were processed with Gen Alex software ver. 3.5 A total of 67 alleles were detected among the different regions analyzed, with an average of 7 alleles for loci ranging from 142 to 240 bp and an effective number of alleles (ENA) of 5.36. The mean heterozygosity was 0.80, and the mean Polymorphic Information Content (PIC) was 0.81. The markers were highly polymorphic; therefore, the most informative SSR primers in the present study would be made up of three markers with PIC, QCA053 (0.87), QCA015 (0.86) and QCA034 (0.86), for determining the genetic identity of Chenopodium quinoa Willd. These markers can be easily interpreted and are useful for the molecular characterization of quinoa varieties. Analysis of the hierarchical clusters using UPGMA (Unweighted Pair Group Method) clustering identified 5 groups at a similarity coefficient of 0.77 among the quinoa varieties studied in this research.