Restriction Endonucleases EcoRI Research Articles

A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases.

All viruses require strategies to inhibit or evade the immunity pathways of cells they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid nucleic-acid targeting immune pathways such as CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) and restriction-modification (R-M) systems to replicate efficiently1. Here, we show that jumbo phage ΦKZ, infecting Pseudomonas aeruginosa, segregates its DNA from immunity nucleases by constructing a proteinaceous nucleus-like compartment. ΦKZ resists many DNA-targeting immune systems in vivo, including two CRISPR-Cas3 subtypes, Cas9, Cas12a, and the restriction enzymes HsdRMS and EcoRI. Cas and restriction enzymes are unable to access the phage DNA throughout the infection, but engineered re-localization of EcoRI inside the compartment enables phage targeting and cell protection. Moreover, ΦKZ is sensitive to the RNA targeting CRISPR-Cas enzyme, Cas13a, likely due to phage mRNA localizing to the cytoplasm. Collectively, we propose that Pseudomonas jumbo phages evade a broad spectrum of DNA-targeting nucleases through the assembly of a protein barrier around their genome.

Haplotipos mitocondriales de Phytophthora andina de tomate de árbol en el Ecuador

Se determinó el haplotipo mitocondrial de 131 aislamientos puros de Phytophthora andina colectados de tomate de árbol (Solanum betaceum) en varias provincias del Ecuador. Se amplificaron las regiones P2 y P4 del ADNmt, y los productos de PCR obtenidos fueron digeridos con las enzimas de restricción MspI y EcoRI. Todos los aislamientos analizados mostraron un patrón de bandas correspondiente al haplotipo Ia para P. infestans, es decir, bandas de tamaño 720 y 350 pb con MspI (región P2) y bandas de tamaño 394, 361 y 309 pb con EcoRI (región P4). Sin embargo, también se observó de forma consistente en todos los aislamientos analizados bandas adicionales que no han sido reportadas anteriormente. Para la región P2 se obtuvieron bandas adicionales de 400 y 500 pb y un fragmento no digerido de 1070 pb, y para la región P4 bandas adicionales de 603 y 755 pb además de un fragmento no digerido de 964 pb. El encontrar un patrón nuevo de bandas de manera consistente para todos los aislamientos de P. andina analizados sugiere que la descripción del haplotipo Ia asignado a P. andina de tomate de árbol deberí­a ser redefinido.

Molecular diversity of hpd gene in clinical isolates of Haemophilus influenzae

Infections due to Haemophilus influenzae result in tremendous global morbidity. The conjugated vaccines against H. influenzae type b (Hib) have dramatically reduced the incidence of invasive Hib disease in the routine immunization of infants. The several proteins used as vaccine candidates for this pathogen, but they don't produce efficient immune in animal models against all strains of H. influenzae. This study aimed to determine the diversity of hpd gene nucleotide sequences of Iranian native clinical isolates of H. influenzae as a native vaccine candidate compared to standard strains.Twenty isolates of H. influenzae recovered from different clinical specimens of patients admitted to Milad and Imam Khomeini hospitals, Tehran, Iran. Then, isolates detected and identified as H. influenzae using biochemical tests, and further confirmation through omp6 gene PCR. The hpd gene was amplified by PCR using gene-specific primers, and the amplicons digested with EcoR1. For four isolates, the Amplicon of hpd gene sequenced, and the sequences aligned with sequences harbored in GenBank. Subsequently, sequences were submitted to the EMBL site (http://www.ebi.ac.uk/embl/).EcoR1 restriction enzyme pattern was the same among the 19 clinical isolates, and only one isolate was different. That different one with 3 out of 19 isolates were sequenced. The results showed that the nucleotide sequences and the deduced amino acid sequences for protein D in clinical isolates were highly conserved with similarities >95%.In conclusion, regarding high similarity up to 99% in clinical isolates, protein D can be a novel vaccine candidate against all types of H. influenza from Iran. This finding should be proved with more isolates, and also, evaluate the immunological features of protein D in animal models.

Fusion recombinant protein expression of newcastle disease virus from Escherichia coli-Cloned C1a using accurapidTM protein expression kit

This research aimed to express F recombinant protein that is clon from genes F of local isolate ND virus which can be used as vaccine candidate in order to improve the effectiveness of ND virus vaccination. Confirmation of NDV pBT7-N-His-Fusion plasmid on C1a clone is done by gel agarose 1 % electrophoresis with staining by using fluorosafe DNA stain. To separate plasmid and insert that contain genes F, cutting is done with EcoRI restriction enzyme. EcoRI enzyme is able to cut NDV pBT7-N-His-Fusion plasmid through 37°C incubation process during three hours. DNA cutting visualization is done by gel agarose 1% electrophoresis by using fluorosafe DNA stain. NDV pBT7-N-His-Fusion plasmid is express by E. coli extract in order to gain F protein. The product of protein expression is visualized by SDS – PAGE and western blot. NDV pBT7-N-His-Fusion plasmid visualization by gel agarose electrophoresis results 4643 bp band. Moreover, from the visualization of Eco RI enzyme cutting on gel agarose electrophoresis result, the researcher found two bands with different size, 4001 bp and 642 bp. After protein expression process 25,6 kDa band is seen both in the result of SDS – PAGE and western blot.

Новый подход к диагностике миодистрофии Ландузи-Дежерина, основанный на полимеразной цепной реакции

Актуальность. Миодистрофия Ландузи-Дежерина (МЛД) является одной из наиболее часто встречающихся мышечных дистрофий. В 95% случаев заболевание связано с частичной делецией массива повторов D4Z4 на одном из аллелей 4-й хромосомы. Существующие диагностические методики гибридизации по Саузерну и молекулярного комбинга являются ресурсо- и времязатратными. В настоящее время в Российской Федерации молекулярно-генетическая диагностика МЛД не проводится. Цель. Поиск новых подходов к диагностике МЛД для использования в молекулярно-генетических лабораториях. Методы. ДНК выделялась в агарозных блоках и подвергалась обработке эндонуклеазой EcoRI. Полученные фрагменты ДНК разделялись методом пульс-электрофореза в агарозном геле, после этого агарозный гель фрагментировался согласно маркеру молекулярного веса и использовался в качестве матрицы для полимеразной цепной реакции (ПЦР). Принадлежность полученных ПЦР-продуктов к последовательностям повторов D4Z4 4-й хромосомы подтверждалась секвенированием по Сэнгеру. Результаты. Протокол пульс-электрофореза оптимизирован таким образом, что после всех этапов ДНК в агарозном геле пригодна для использования в качестве матрицы для ПЦР. Разработана ПЦР-система специфичной амплификации контрольных ДНК-матриц 4-й хромосомы и подтверждена секвенированием принадлежность получаемых ПЦР-продуктов к последовательности повторов D4Z4 4-й хромосомы. Выводы. Показана возможность использования ДНК в агарозном геле после пульс-электрофореза в качестве матрицы для детекции повторов D4Z4 методом ПЦР. Представленная ПЦР-система специфично амплифицирует последовательности D4Z4 4-й хромосомы. Используя данную ПЦР-систему и геномную ДНК пациента с известной длиной массива повторов D4Z4 проведена успешная диагностика МЛД. Таким образом разработан новый подход к диагностике МЛД для использования в молекулярно-генетических лабораториях. Relevance. Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies. In 95% of cases, the disease is associated with partial deletion of the array of the D4Z4 repeats on one of the alleles of the 4th chromosome. The existing diagnostic methods of Southern blotting and molecular combing are quite resource-and time-consuming. At the moment, molecular genetic diagnostic of FSHD is not provided on the territory of the Russian Federation. Aim: to find new approaches for molecular genetic diagnostic of FSHD acceptable for use in standard molecular genetic laboratories Materials and methods: DNA isolated in agarose plugs and treated by the EcoRI restriction enzyme. DNA fragments then were separated by pulse field gel electrophoresis (PFGE) in agarose gel. After PFGE, the agarose gel was fragmented and used as a matrix for PCR. The identity of the obtained PCR products to the sequence of the D4Z4 repeats of the 4th chromosome was confirmed by sequencing by Sanger. Results. The PFGE protocol is optimized in such a way that, after all stages, DNA in agarose gel is suitable for use as a matrix for PCR. We achieve a specific amplification of the control DNA matrices of the 4th chromosome and confirm belonging of the PCR products to the sequence of D4Z4 repeats of the 4th chromosome by the Senger sequencing. Conclusions. This paper shows the possibility of using DNA in agarose gel after PFGE as a matrix for detection of D4Z4 repeats by PCR. The presented PCR system specifically amplify sequence of the 4th chromosome D4Z4 repeats. Using this PCR system and genomic DNA of a patient with a known length of the D4Z4 repeats array, a successful diagnosis of FSHD was performed. Thus, we propose a new approach for FSHD diagnostic, acceptable for use in standard molecular genetic laboratories.

Molecular characterization of phytoplasma diseases of pepper in Turkey

AbstractDuring autumn, an extensive survey was conducted in pepper (Capsicum annum L.) in intensive cultivation areas of four provinces in southeastern Turkey (Adana, Kahramanmaraş, Mersin and Şanlıurfa) in order to identify the causal agent (s) of phytoplasma‐like symptoms (chlorosis, little‐leaf, short internodes and stunting). DNA amplification by PCR and RFLP analysis using EcoRI restriction enzyme confirmed the presence of phytoplasmas in Şanlıurfa and Mersin, and consequently their possible association with the symptoms. Sequencing and phylogenetic analysis revealed that the isolate from Şanlıurfa had 99% sequence identity with “Candidatus Phytoplasma trifolii” (16SrVI) and is a member of the clover proliferation group (16SrVI‐A). Additionally, the isolate from Mersin had 96% sequence identity with “Candidatus Phytoplasma asteris” (16SrI). Importantly, gene sequence of the Mersin isolate shared <97.5% similarity to previously discovered “Ca. Phytoplasma” species. Consequently, the phytoplasma detected from Mersin could represent a new “Ca. Phytoplasma” species and to our knowledge, this is the first report of asteris‐like phytoplasmas infecting pepper in Turkey.

Enhanced solid phase extraction of DNA using hydrophilic monodisperse poly(methacrylic acid-co-ethylene dimethacrylate) microparticles.

The efficiency of solid phase extraction (SPE) of DNA on polymer particles is limited by the features of the applied solid support, such as size, hydrophilicity, and functionality and their application in SPE also requires additional steps and compounds to finally obtain sufficient amount of high-quality DNA. The present study describes a preparation of sub-micrometer monodisperse poly(methacrylic acid-co-ethylene dimethacrylate) (PME) particles by precipitation polymerization. The effect of the ethylene dimethacrylate (EDMA) crosslinker concentration on morphology and particle size, which varied from 730 to 900nm, was investigated. The particles with 5 and 15wt% EDMA were selected for a study of SPE of plasmid DNA under various adsorption and elution conditions, followed by the enzymatic restriction of isolated DNA to verify a quality the nucleic acid. The particles with 15wt% EDMA were suitable for the SPE because they retained better colloidal stability during the adsorption without additional induction of DNA conformational change. The quality of isolated DNA was finally verified by enzymatic restriction by restriction endonuclease EcoRI. Moreover, the developed method using PME particles was successfully utilized for DNA isolation from Escherichia coli lysate.

Periplasmic expression of a restriction endonuclease in Escherichia coli and its effect on the antiviral activity of the host

One possible mechanism preventing phage infection of the bacterial cells is related to the presence of an effective restriction-modification system (R-M) which allows restriction of the invading DNA. However, there are some limitations to the absolute restriction of foreign DNA. Since there is a serious conflict between increase in the restriction-modification genes expression level and cell viability, we examined the antiviral effect of EcoRI restriction endonuclease after its translocation to the periplasmic space of the cell. We assumed that such reconstructed R-M system could be able to degrade foreign DNA at the stage of its passage through the cell envelope of Gram-negative bacteria, before its penetration into the bacterial cytoplasm. The Tat secretion pathway of Escherichia coli was used to export R.EcoRI fused to the TorA leader peptide across the cytoplasmic membrane. However, although we observed a huge accumulation of the TorAss-R.EcoRI pre-protein in the cytoplasm the Tat system did not provide an efficient transport across the cytoplasmic membrane. Moreover, our data strongly suggest that endonuclease cannot function under the conditions prevailing in periplasmic space, therefore, the transported endonuclease could not contribute to an increase in restriction properties of the host.

The Production of the Recombinant Protein of Anthrax Bacteriolysine PlyPH and In Vitro Study of the Lytic Properties of the Protein Coded for them Against Bacillus anhracis Microbial Cells

The causative agent of anthrax - Bacillus anthracis, due to the prevalence of its natural foci in Russia, high virulence for humans and most mammals, the unique resistance of spore forms to environmental factors and repeated use in terrorist acts, is an extremely dangerous biological agent. Therefore, the search for new effective drugs for the diagnosis and treatment of anthrax, including diseases caused by antibiotic-resistant strains of B. anthracis is necessary. The use of lytic enzymes of species-specific bacteriophages is a new trend in the diagnosis, prevention and treatment of infectious diseases. The goal of this work is the cloning of the anthrax bacteriolysin PlyPH gene as part of the pTrcHis2C vector in Escherichia coli and the in vitro study of the lytic properties of the protein encoded by it against B. anthracis microbial cells. According to the complete sequencing of the B. anthracis genomes of the Ames, Stern 34F2 and JB17 strains, a prophage was found in their chromosomal DNA, which lost part of the structural genes necessary for its replication, but retained a gene with a high degree of homology with the bacteriolysin γ phage gene. For amplification and subsequent cloning of the PlyPH gene, we developed primers containing EcoRI and BamHI restriction enzyme recognition sites. Amplification of the PlyPH gene in a polymerase chain reaction (PCR) with a developed pair of primers was performed using the Stern 34F2 strain of the anthrax microbe as a template. Based on the obtained amplification products and the pTrcHis2C vector, we constructed a recombinant plasmid containing the bacteriolysin synthesis PlyPH gene and stably functioning in the cells of the recombinant E. coli strain. In the course of research, it has been established that microbial cells of the E. coli recombinant TOP10 strain provide for the production of the bacteriolysin of the anthrax prophage, PlyPH , which has the ability to in vitro lyse the vegetative cells of the STI-1 vaccine strain of B. anthracis

Designing and construction of a DNA vaccine encoding ag85a and tb10.4 fusion genes of Mycobacterium tuberculosis

Background and Aim : Novel TB vaccines that aim to boost and/or replace Bacillus Calmette-Guerin (BCG) are currently in development. DNA vaccines can stimulate both humoral and cell-mediated immunity in different animal models of TB and is thought to be a promising strategy in the development of new vaccines against TB. The aim of this study was to design and construct a DNA vaccine encoding ag85a and tb10.4 fusion genes of Mycobacterium tuberculosis . Materials and Methods: Tb10.4 fragment was amplified by PCR and the products were digested with restriction enzymes. Next, it was cloned into the pcDNA3.1 + plasmid. The ag85a gene and pcDNA3.1 + / tb10.4 plasmid were digested by EcoRI and BamH1 restriction enzymes. Eukaryotic cells were transfected with pcDNA3.1 + / tb10.4 - ag85a plasmid for confirming expression of tb10.4 - ag85a in these cells. Results: Using electrophoresis of PCR products, fragments 297 bp for tb10.4 and 1017 bp for ag85a were observed. Eukaryotic cells transfection with pcDNA3.1 + / tb10.4 - ag85a vector was confirmed with cDNA synthesis and existence of tb10.4-ag85a was confirmed with RT-PCR. Conclusion: In this study, we constructed a DNA vaccine encoding tb10.4-ag85a fusion fragment. It can be used for development of more new DNA vaccines in future studies .

Molecular characterization of Aspergilli isolated from outdoor air

Ubiquitous airborne conidia of the genus Aspergillus are responsible for a diverse group of human disorders from allergy to life treating invasive aspergillosis and mycotoxicoses. The aim of this study was to determine the population structure of Aspergillus isolated from outdoor air in Tehran by comparing the nucleotide sequences of ITS region and the PCR-RFLP molecular method. Internal transcribed spacer domains of 47 Aspergillus spp. were amplified and sequenced and PCR products were digested individually with restriction enzymes TaqI and EcoRI. For all species the PCR reaction produced a fragment of approximately 600bp in length. All of the nucleotide sequences were highly similar with the corresponding reference sequences registered at the gene bank. The all isolates displayed same banding pattern on the basis EcoR1 cleavage. While Taq1 enzyme profiling provided 5 different banding pattern. The results show that the A. niger section has the highest frequency with 27 isolates (57.4%). Of these, 23 isolates (48.9%) belonged to the A. niger complex and 4 isolates (8.5%) to the A. aculeatus complex. The A. flavus complex was also placed in the next ranking with 9 isolates (19.1%). These results strongly support the need for using molecular markers as an auxiliary tool in differentiating Aspergillus species.

PROTOTYPE DESIGN OF LYMPHOCYTE TCD4+ RESISTANT AGAINST HIV INFECTION GENERATED FROM PERIPHERAL BLOOD HAEMATOPOIETIC STEM CELL (PBMCs) By DELETION OF 32 bp CCR5 ENCODING GENE

AIDS epidemic has spread to all parts of Indonesia and currently more than 150 countries reported the existence of HIV/AIDS from around the world. Additionally, HIV/AIDS treatment using ARV drugs also find obstacles that must be faced in terms of host, environment and the agent. The objective of this study was to generate lymphocytes TCD4+ that are resistant to HIV infection generate from PBMCs through by deletion of 32 bp CCR5 encoding gene. In principle, this study was done in three steps. First, isolation, culture and purification of lymphocyte TCD4+ from PBMC (Mather, 2008; Rantam, et.al., 2009). Second, lymphocyte TCD4+ characterization by PCR with primer F 5’CAAGTCGAGCGCCCCGCAAGGGG-3, R 5’GTCCGAGTGTGGCTGATCATCC-3 (Thomsen, et.al., 2002; Yuwono, 2006; Hall and Ziedonis 2007; Purwati, et.al., 2009). Third, designing of lymphocyte TCD4+ prototype which was resistant to HIV infection by deletion of 32 bp CCR5 full gene. Results: Twenty-four hours after culture, there were abundant cell growths. TCD4+ lymphocytes from isolated and cultured 10 ml PBMC were found to be 2 x 107. Phenotype characterization of TCD4+ lymphocyte provided positive results, while the genotype showed similarities to that in corresponding gene bank of CCR5 variant A and variant B. Prototype of HIV resistant TCD4+ lymphocytes was made by nucleotide deletions in conserved areas, at position 554-576 bp, using restriction enzymes EcoRI checked using PCR and sequencing. In conclusion, prototype design of HIV resitent TCD4+ lymphocytes is obtained through the deletion of 32 bp CCR5 encoding full gene at GTCAGTATCAATTCTGGAA GAATTT CCAGACA using EcoRI enzyme.Keywords: HIV/AIDS, resistant TCD4+ lymphocytes, mutant 32 bp CCR5, PBMCs, deletion

Simple and efficient genomic DNA extraction protocol for molecular characterisation of Phytophthora colocasiae causing taro leaf blight

Genomic DNA extraction protocol with relatively high quantity and purity is prerequisite for the successful molecular identification and characterisation of plant pathogens. Conventional DNA extraction methods are often time-consuming and yield only very poor quantity of genomic DNA for samples with higher mycelial age. In our laboratory, we have aimed at establishing an efficient DNA isolation procedure, exclusively for the oomycete pathogen Phytophthora colocasiae causing serious leaf blight disease in taro. For this a phenol free protocol was adopted, which involves SDS/Proteinase K-based inactivation of protein contaminants, extraction of nucleic acids using chloroform: isoamyl alcohol and later precipitation of genomic DNA using isopropanol and sodium acetate. The purity of the isolated DNA was analysed by A260/280 and A260/230 spectrophotometric readings and confirmed by restriction digestion with restriction enzyme Eco RI. In this study, a comparative assessment was done with CTAB method and the commercial genomic DNA purification kit (Thermo Fisher Scientific, Fermentas, EU). The extracted DNA was found to be suitable for further downstream applications like ITS amplification of the rDNA ITS region and PCR amplification with species-specific primers.

Antibiotic resistance and distribution of SodCI, sopE, sefA genes among Salmonella enteric serotype Enteritidis isolates from poultry

Present work aimed to examine the antibiotic resistance of the Salmonella enterica serotype Enteritidis (SE) isolates from poultry, to study the plasmid-mediated ampicillin resistance and to detect and determine the distribution of sodCI, sopE and sefA genes. Thirty-five SE isolates from one-day chicks, layers and broilers were studied for susceptibility/resistance to sixteen antimicrobial agents; 23 (65.7%) of them showed resistance to ampicillin, 5 (14.3%) to ampicillin and tetracycline, 4 (11.45%) to tetracycline and 1 (2.9%) isolate showed multi-drug resistance. Ampicillin (AmpR) and ampicillin/tetracycline (AmpRTeR) resistance was easily transferred by conjugation, and all isolates except two possessed a common band. The molecular mass of the plasmid carrying ampicillin resistance was approximately determined at 41kb after DNA digestion with BamHI, HindIII, EcoRI, EcoRV and PstI restriction enzymes and ligation of EcoRI fragments to pET29c. For the detection of TEM-1 or/and TEM-2 ß-lactamases, two pairs of primers were used in a polymerase chain reaction (PCR). The PCR products showed the presence of blaTEM-1 gene in all isolates. The presence of SodCI, sopE, sefA genes was also examined by PCR. Twenty-two (62,8%) isolates carried the sodCI gene, thirty-four (97,2%) isolates carried the sopE gene and all isolates carried the sefA fimbrial locus.

Isolation, cloning and molecular analysis of ag85a and tb10.4 genes from Mycobacterium tuberculosis

Background: Novel tuberculosis (TB) vaccines that aim to boost and/or replace Bacillus Calmette-Guerin (BCG) are currently in development. DNA vaccines can stimulate both humoral and cell-mediated immunity in different animal models of TB and is thought to be a promising strategy in the development of new vaccines against TB. The aim of this study was to design and construct a DNA vaccine encoding ag85a and tb10.4 fusion genes of Mycobacterium tuberculosis. Materials and Methods: tb10.4 fragment was amplified by PCR and the product was digested with restriction enzymes. Next, it was cloned into the pcDNA3.1+ plasmid. The ag85a gene and pcDNA3.1+/tb10.4 plasmid were digested by EcoRI and BamHI restriction enzymes. Constructed vector was sequenced. The molecular analysis was done using bioinformatics software. New chimeric vector containing ag85a-tb10.4 genes were purified. Expression of pcDNA3.1+/tb10.4-ag85a plasmid was confirmed in eukaryotic cells. Results: Fragments of 297 bp for tb10.4 and 1017 bp for ag85a were observed in agarose gel electrophoresis. Alignment of ag85a-tb10.4 genome sequence with reference genes in GenBank showed exact identities that indicate correction of all cloning procedures. Transfection of eukaryotic cells with pcDNA3.1+/tb10.4-ag85a vector and existence of tb10.4-ag85a fusion gene were both confirmed with RT-PCR. Conclusion: In this study, tb10.4 and ag85a genes were isolated from Mycobacterium tuberculosis H37Rv strain and cloned into pcDNA3.1+. Also, the capability of constructed vector in producing fusion ag85a-tb10.4 protein was confirmed with RT-PCR. pcDNA3.1+/tb10.4-ag85a vector can be used for further studies in future. K e y w o r ds: Mycobacterium tuberculosis ; ag85a; tb10.4; pcDNA3.1+; DNA vaccine.

Detection of Russian olive witches’-broom disease and its insect vector in Northwestern Iran

Abstract Recently, Russian olive trees showing witches’-broom and little leaf symptoms have been widely observed in northwestern and central Iran. Polymerase chain reaction (PCR) and nested PCR assays using phytoplasma universal primer pairs confirmed phytoplasma symptomatic infection of trees. Sequence analyses showed that ‘Candidatus Phytoplasma asteris’ was the causal agent of the disease in these regions. However, RFLP results using restriction enzymes HpaII, EcoRI, HinfI and AluI indicated that the collected isolates in these regions are genetically different. In addition, leafhopper Macropsis infuscata was recognized as a possible insect vector of the disease for the first time.

Agrobacterium tumefaciens -mediated transformation of tomato (Solanum lycopersicum)

The present investigation describes the development of genetically engineered tomato plants with annexin gene. The alkaline lysis method is used to isolate the plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) from E.coli (DH5α strain). The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

METODE EKSTRAKSI DNA CABAI (<em>Capsicum annuum</em> L.) MENGGUNAKAN MODIFIKASI BUFFER CTAB (CETHYL TRIMETHYL AMMONIUM BROMIDE) TANPA NITROGEN CAIR

Chili pepper is an agricultural commodity having high economic value. The production and supply of chili pepper frequently did not match the increased demand; it caused the market price fluctuated. It is important to create new varieties of chili pepper with high production trait to overcome the scarcity. Therefore the plant breeding activities for chili pepper should be done intensively in both conventional and molecular-based to obtain varieties of chili pepper with expected qualities. In molecular breeding, DNA extraction is the crucial steps of the process. If extracted DNA has an excellent quality and quantity, the next processes normally could be completed with the high-quality result. To date, most methods of DNA extraction used liquid nitrogen to destroy the tough carbohydrates of plant tissue. Liquid nitrogen is nitrogen gas in a fluid state which quite difficult to be distributed to the remote laboratory wit no available storage facility. This study aimed to obtain a modified DNA extraction method, in particular for chili pepper, which capable to produce DNA with high quality and quantity without using liquid nitrogen. The sample used consisted of eight F2 plants including their hybrid-parental of the Kencana and the 0207. This research applied modified Doyle and Doyle method for extraction. Modification of extraction buffer is done through the addition of the 1% (w/v) PVP (Polyvinylpyrrolidone) and 0.2% (v/v) β-mercaptoethanol. The results showed that the DNA extracted using this method has good quality and quantity, capable of being amplified by using SSR (Simple Sequence Repeat) primer and could be digested by restriction enzyme EcoRI. Besides, this method can reduce dependence on the use of liquid nitrogen, in particular for remote laboratories with no available storage facility.

Agrobacterium tumefaciens -mediated transformation of tobacco (Nicotiana tabaccum)

The present study involves the development of genetically engineered tobacco plants with annexin gene. The plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) were isolated from E.coli (DH5α strain) by alkaline lysis method. The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

Determination of restriction enzyme activity when cutting DNA labeled with the TOTO dye family

ABSTRACTOptical mapping, a single DNA molecule genome analysis platform that can determine methylation profiles, uses fluorescently labeled DNA molecules that are elongated on the surface and digested with a restriction enzyme to produce a barcode of that molecule. Understanding how the cyanine fluorochromes affect enzyme activity can lead to other fluorochromes used in the optical mapping system. The effects of restriction digestion on fluorochrome labeled DNA (Ethidium Bromide, DAPI, H33258, EthD-1, TOTO-1) have been analyzed previously. However, TOTO-1 is a part of a family of cyanine fluorochromes (YOYO-1, TOTO-1, BOBO-1, POPO-1, YOYO-3, TOTO-3, BOBO-3, and POPO-3) and the rest of the fluorochromes have not been examined in terms of their effects on restriction digestion. In order to determine if the other dyes in the TOTO-1 family inhibit restriction enzymes in the same way as TOTO-1, lambda DNA was stained with a dye from the TOTO family and digested. The restriction enzyme activity in regards to each dye, as well as each restriction enzyme, was compared to determine the extent of digestion. YOYO-1, TOTO-1, and POPO-1 fluorochromes inhibited ScaI-HF, PmlI, and EcoRI restriction enzymes. Additionally, the mobility of labeled DNA fragments in an agarose gel changed depending on which dye was intercalated.