Set Of Restriction Enzymes Research Articles

Here and there: the double-side transgene localization.

Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identif ication of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact f iltration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene’s f lanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two f lanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the f ield of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method’s feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to eff iciently localize genomic transposon integrations, which was conf irmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.

Restriction site-associated DNA from Python-implemented Digestion Simulations (RApyDS): a companion tool for RAD sequencing experimental design

Reduced representation sequencing is a practical approach for obtaining genetic variations from a random subsample of the genome. RADseq (Restriction Site-Associated DNA Sequencing), as one of the more popular reduced representation approaches, is currently being used in a wide array of applications including marker development, phylogenetics, and population genomics. A crucial step in designing a RADseq experiment is the selection of one or a pair of restriction enzymes (RE) that will result in sufficient density of loci to meet the objectives of the study, which is not straightforward because of difficulties in obtaining a standard set of REs that can generally be applied to RADseq experimental designs. Here we present RApyDS, a simulation tool that provides users with evaluation metrics to aid in choosing suitable REs based on their target RADseq design. RApyDS can perform simulations for single- or double-digest RADseq, preferably with a supplied reference genome. The tool outputs an overview page, electrophoresis visualization, mapping of restriction cut sites, and RAD loci density across the genome. If supplied with an annotation file, the program can also output evaluation metrics for a specified genomic feature. The tool is currently available at https://github.com/pgcbioinfo/rapyds.

DETECTION OF POSPIVIROIDS IN THE CZECH REPUBLIC AND THEIR DISCRIMINATION BY RESTRICTION ANALYSIS

Potato spindle tuber viroid (PSTVd), a member of the genus Pospiviroid, is a quarantine pest in the European Union and its presence in ornamental solanaceous crops represents a serious threat to susceptible vegetable and potato crops. Since the year 2007 the Czech Plant Protection Service (PPS) carries out a survey for PSTVd on ornamental solanaceous species. Within the first year of the survey, several new hosts of PSTVd were identified. Also, a new species of Pospiviroid was detected in the Czech Republic. At the beginning of the survey, a method for detection of PSTVd recommended in the EPPO diagnostic protocol was adopted. Reverse transcription-polymerase chain reaction with a semi-specific primer pair was carried out in the diagnostic laboratory of PPS while the sequencing of PCR products was performed in an external laboratory. Due to this time consuming process, which proved inefficient for the needs of rapid application of phytosanitary measures, the laboratory developed a faster procedure. The entire genomic sequences of all viroids which contained primer identical segments were downloaded from the GenBank database and aligned to search for viroid-specific restriction sites. A combination of four enzymes was chosen to distinguish the detected viroids. Restriction analysis was successfully tested and applied for the PSTVd identification. Later on, after confirmation of other pospiviroids detected for the first time in the Czech Republic (Tomato apical stunt viroid and Chrysanthemum stunt viroid on Solanum jasminoides and Citrus exocortis viroid on Solanum jasminoides and Verbena A— hybrid), the diagnostic method was improved to detect a wide range of Pospiviroid species with the introduction of primers detecting the five most common pospiviroids in the ornamental solanaceous plants in Europe. Similarly, for identification of the amplified PCR products, a new set of restriction enzymes was identified.

Differentiation of lactic acid bacteria based on RFLP analysis of the tuf gene

The tuf gene, which encodes the elongation factor Tu, is universally distributed in Gram-positive bacteria. A small region of the tuf gene was evaluated for the differentiation of Lactobacillus strains using restriction fragment length polymorphism (RFLP) analysis. The 803 bp portion of tuf genes from 9 closely-related Lactobacillus reference strains were successfully amplified by polymerase chain reaction (PCR), and restriction patterns of each were evaluated with a set of restriction enzymes, which included AluI, HaeIII, and Sau3AI. The results implied that restriction patterns obtained from 2 of these restriction enzymes (AluI and HaeIII) could effectively differentiate closely-related Lactobacillus species.

Isolation of Chromatin DNA Tightly Bound to the Nuclear Envelope of HeLa Cells

Recent discovery of the role of nuclear pores in transcription, predicted by our early DNA-membrane complex (DMC) model, makes membrane-bound DNA (MBD) isolation from the cell nucleus and analysis of the MBD actual. The method of MBD isolation proposed by us retains DMC integrity during isolation. We used HeLa cells for DMC extraction. Changing the ionic composition of the isolation medium and replacing DNase I, used commonly for chromatin destruction, with a set of restriction enzymes allowed us to isolate the MBD. Treatment of a nuclear membrane with proteinase K and ultrasound has been used to increase the yield of MBD. Electron microscopic analysis of the purified fraction of isolated DMC supports our previous model of nuclear envelope lipid-chromatin interaction in the nuclear pore assembly.

HCV-Core Region: Its Significance in HCV-Genotyping and Type Dependent Genomic Expression

Present study reports the standardization and use of polymerase chain reaction (PCR) followed by RFLP (PCR-RFLP) implicating HCV-core as a target region and its comparison with PCR-RFLP based on the use of 5'NCR for HCV genotyping / sub-typing. The new assay produced 399bp PCR product from core region and used a different set of restriction enzymes (MboI, AccI and BstNI) to digest these products for HCV sub-typing. This assay allows a quick determination of HCV types / sub-types. HCV was typed / sub-typed using these two assays in patients with liver and renal diseases. Analysis of results demonstrated the two assay system to be comparable with some difference in few cases. Whereas genotype-3 was a common finding in liver disease groups, genotype-1 was more frequent in chronic renal failure cases. The PCR product from core based assays was also subjected to sequence analysis for genotypes/sub-types and the findings were slightly different. This was further analysed by phylogenetic analysis which supported our findings.

Genetic Diversity of Sugarcane Grassy Shoot (SCGS)-Phytoplasmas Causing Grassy Shoot Disease in India

Sugarcane grassy shoot (SCGS) caused by SCGS-phytoplasmas is a serious disease of sugarcane. Since the diseased plants exhibit various phenotypic symptoms under field conditions, diagnosis of the disease becomes difficult. A detailed study was conducted on validating phytoplasma specific primers for the diagnosis of the disease and to identify the extent of variations in rDNA genome of SCGS-phytoplasmas. A total of 37 sugarcane samples with different phenotypic symptoms of SCGS were collected from various states of India. Nested-PCR amplification using two sets of forward (P1/P7) and reverse primers (P4/P7) of 16S rRNA gene, 16S–23S rRNA spacer region (SR) and 23S rRNA gene sequences amplified SCGS-phytoplasmas in all the suspected samples. Nucleotide sequence analysis clearly revealed that 18 isolates of SCGS-phytoplasmas from different locations/varieties showed no variation in the targeted gene sequences. Although there were significant variations in phenotypic expression of SCGS phytoplasmas on sugarcane, we could not establish any genotypic variation among the pathogenic isolates in rDNA region. All our sequences matched exactly with other reported phytoplasmal isolates of SCGS-Pun (DQ459439), SCGS (DQ380341), SCGS-CM1 (AM269740) and SCGS-SV1 (AM269742) from India. Among the other reported SCGS phytoplasmas it shared sequence similarity ranging from 99.7 to 94.3%. Our SCGS phytoplasmas showed 99.6% similarity with other phytoplasmas infecting sugarcane such as sugarcane white leaf (SCWL) and 97.8% with SCYL. The closely related phytoplasmas such as BraWL and SGS phytoplasmas showed 99.6% similarities with our SCGS-phytoplasmas. However, distantly related phytoplasmas FBP and CBY1 and StLL showed sequence similarities between 84.3 and 82.4%. Restriction of the amplicons with a set of restriction enzymes did not show any polymorphism among them. 16S rRNA, 16S–23S rRNA SR and 23S rRNA sequencing and restriction fragment length polymorphism (RFLP) results proved that the phytoplasma isolates infecting sugarcane in India with phenotypic variations from different regions have similar ITS region and further studies are needed to locate other genes in phytoplasma genome which determine variations in phenotypic expression in the SCGS disease.

CLASSIFICATION AND IDENTIFICATION OF FUNGAL SEQUENCES USING CHARACTERISTIC RESTRICTION ENDONUCLEASE CUT ORDER

Restriction Fragment Length Polymorphism (RFLP) is a powerful molecular tool that is extensively used in the molecular fingerprinting and epidemiological studies of microorganisms. In a wet-lab setting, the DNA is cut with one or more restriction enzymes and subjected to gel electrophoresis to obtain signature fragment patterns, which is utilized in the classification and identification of organisms. This wet-lab approach may not be practical when the experimental data set includes a large number of genetic sequences and a wide pool of restriction enzymes to choose from. In this study, we introduce a novel concept of Enzyme Cut Order - a biological property-based characteristic of DNA sequences which can be defined and analyzed computationally without any alignment algorithm. In this alignment-free approach, a similarity matrix is developed based on the pairwise Longest Common Subsequences (LCS) of the Enzyme Cut Orders. The choice of an ideal set of restriction enzymes used for analysis is augmented by using genetic algorithms. The results obtained from this approach using internal transcribed spacer regions of rDNA from fungi as the target sequence show that the phylogenetically-related organisms form a single cluster and successful grouping of phylogenetically close or distant organisms is dependent on the choice of restriction enzymes used in the analysis. Additionally, comparison of trees obtained with this alignment-free and the legacy method revealed highly similar tree topologies. This novel alignment-free method, which utilizes the Enzyme Cut Order and restriction enzyme profile, is a reliable alternative to local or global alignment-based classification and identification of organisms.

Rapid identification of lactic acid bacteria isolated from home-made fermented milk in Tibet

Fermented milk is a very delicious and nutritional food distributed in the international markets. Rapid preliminary identification of lactic acid bacteria to the species level is an important issue for the fermentation industry. One hundred seventy-one strains of lactic acid bacteria (LAB) were isolated from twenty-nine kurut samples and fifteen traditional fermented mongolian cattle milk (FMCM) samples in Tibet, China. All of the strains were isolated and divided into bacilli or cocci by phenotype, and then differentiated by restriction fragment length polymorphism (RFLP) analysis using a set of restriction enzymes, AluI, HaeIII, BsmaI, TspRI and HinfI. Restriction pattern analyses indicated that the lactobacilli could be clearly identified at the species level and the LAB cocci at the genera level by the five restriction enzymes. Further studies on 16S rDNA sequences of representative and undefined strains showed that the combination of RFLP analysis of polymerase chain reaction (PCR) for amplifying 16S rDNA and 16S rDNA sequence analysis is rapid, easy to perform, and effective for large-scale preliminary identification of LAB.

Development of a Simple PCR-based Marker for the Determination of Growth Habit in Vicia faba L. using a Candidate Gene Approach

We have developed the first molecular marker suitable for the selection of determinate growth habit in faba bean using the candidate gene approach. We obtained the sequences of TFL1/CEN like genes from public databases and designed primers on conserved domains. We used three cultivars with determinate growth habit and four accessions (two cultivars and two lines) with indeterminate growth habit. All these genotypes are used in our faba bean breeding program. A single monomorphic PCR fragment was obtained. A set of restriction enzymes was assayed. The enzyme Hind1II produced a clear polymorphism between determinate and indeterminate genotypes. This new cleaved amplified polymorphism (CAP) marker was tested using an F2 population contrasting for growth habit derived from the cross ‘Verde Bonita’ × 2N52. This marker showed 100% efficiency in discriminating both types of genotypes. Moreover, the codominancy of this marker allows the detection of heterozygous individuals facilitating the breeding process when pyramiding different genes. The perfect cosegregation of the marker with the trait indicates that an orthologue of TFL1/CEN controls the growth habit in faba bean. This marker has been tested in all the genotypes used in our faba bean breeding program as donors of the determinate growth habit. Therefore, it is expected to work well in all the crosses performed with these parental lines as happens in the F2 tested. The CAPS marker developed in this work will be useful for Marker Assisted Selection programs. In addition, this marker is useful for quality control to determinate the percentage of outsider seeds in commercial seed lots. Moreover, it is a valuable tool to breeders when submitting new faba bean varieties for registration since the method allows guaranteeing that outsider plants remains under the requested limit for registration.

RFLP of 16S-ITSrDNA Region to Differentiate Lactobacilli at Species Level

The 16S-ITS (internal transcribed spacer) region of the rrn operon was amplified by polymerase chain reaction (PCR). The amplification products were analysed by restriction fragment length polymorphism (RFLP) using a set of restriction enzymes, AluI, HaeIII, and TaqI. Restriction pattern analyses revealed that TaqI restriction enzyme could clearly differentiate the nine reference strains of Lactobacillus used in the study.

Novel family shuffling methods for the in vitro evolution of enzymes.

It has recently been shown that shuffling of the amino acid sequences of family enzymes allows the generation of improved enzymes. Family shuffling is generally achieved by a DNase I treatment and then by PCR. Shuffling of the xylE and nahH genes, both encoding catechol 2,3-dioxygenases, was carried out by the published method. However, nahH-xylE hybrids were only formed at a very low frequency (less than 1%). Therefore, we developed improved methods for family shuffling by which DNA was cleaved by restriction enzymes instead of by DNase I. With the first improved method, five nahH fragments and five xylE fragments that had been generated by restriction enzyme digestion were subjected to the PCR reactions in two steps, the first being without a primer and the second with a set of primers. This method enabled nahH-xylE hybrid genes to be formed at a high frequency (almost 100%). With the second improved method, nahH and xylE were cleaved by several sets of restriction enzymes, and these digests were then reassembled in two steps. The nahH and xylE DNAs were each cleaved by two (or three) sets of restriction enzymes, and one type of nahH digest and one type of xylE digest were mixed, thus making four (or nine) different mixtures of the nahH and xylE digests. These mixtures were used as templates to carry out PCR without a primer. After the first PCR reaction, all the mixtures were combined, and a second PCR reaction was carried out without a primer. Following these two PCR assembly steps, a third PCR reaction was carried out with two primers to amplify the full-length nahH-xylE hybrid genes. This second method also yielded nahH-xylE hybrids at a frequency of 100%. The degree of recombination of the products with the second method was higher than that with the first method. These methods were used to isolate catechol 2,3-dioxygenases exhibiting relatively high stability at high temperature, one of them being respectively 13- and 26-fold more thermostable than XylE and NahH at 50 degrees C.

Isolation and characterization of a cryptic plasmid from Erwinia citreus ATCC 31623.

A multiple-copy plasmid pPZG500 (3.8 kb) was isolated from a phytopathogenic bacterium Erwinia citreus ATCC 31623. This is the smallest plasmid so far isolated from the genus Erwinia. The plasmid was partially characterized by a set of restriction enzymes and the unique restriction sites were mapped for HindIII, EcoRI, EcoRV and XBaI, while three sites were found for BglII. Nineteen other enzymes did not cut pPZG500. By deletion analyses minimal regions required for replication (ori) and segregational stability (par) were localized on 1.4 kb EcoRV/BglII and 0.7 kb Bgl/II/EcoRI fragments, respectively. The erythromycin resistance marker (Emr) was cloned into pPZG500 and two plasmid derivatives, pPZG502 and pPZG503, were constructed expressing erythromycin resistance as a good selective marker for recombinant selection in Erw. citreus and Escherichia coli. The segregational stability of both constructed plasmids during 90 generations in E. coli JM109 and Erw. citreus C-4 showed that plasmid pPZG503 lacking the presumptive par region was lost from the population at a higher rate. The results of this study demonstrate that plasmid pPZG500 and derivatives are suitable prerequisites for the construction of useful cloning vector(s) in the genus Erwinia.

Direct sequencing of double-stranded PCR products with the sequenase kit and [alpha-35S] dATP.

The polymerase chain reaction (PCR) products are double-stranded linear DNA molecules. Although digestion with a set of restriction enzymes, hybridization with an internal probe, detection of a single-stranded chain polymorphism, or of a site for specific chemical cleavage may all provide useful information on the amplified products, clear-cut identification of nucleic acids is best achieved by sequencing. When a PCR fragment is heterogeneous, cloning in a vector may be required for sequencing each individual molecule independently. In many cases, however, regions of or often the entire PCR product is homogeneous, and direct sequencing without cloning may be undertaken. We have developed a simple and fast method for directly sequencing linear double-stranded DNA molecules, such as PCR products (), which we describe in detail below.

Construction of D29 shuttle phasmids and luciferase reporter phages for detection of mycobacteria

Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacteriophage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria.

Polymerase chain reaction amplification and restriction enzyme typing as an accurate and simple way to detect and identify human papillomaviruses

A simple and economic method for the detection and identification of human papillomaviruses (HPV) is described. The method has been developed with cloned HPV DNA and DNA from clinical samples. Genomic fragments were obtained from several different HPV types, including the ones most frequently encountered in the genital tract by polymerase chain reaction (PCR) amplification directed by degenerate general primers. The amplification fragments were identified by a form of miniature fingerprinting, with a set of restriction enzymes that gave a unique digestion pattern for each HPV type. Different strategies are proposed, based on PCR and restriction analysis, and this approach to identification was compared with more classic methods such as Southern hybridisation.

Xenotropic virus-related restriction patterns of non-H-2 histocompatibility mutant mice strains.

The relationship between alteration in the number of xenotropic virus-related sequences and non-H-2 histocompatibility (H) mutations in mice was investigated. Mutant classifications included gain, loss, and loss-gain mutations. Genomic DNA from a panel of non-H-2 H mutant strains on the C57BL/6 and BALB/c backgrounds was digested with a set of restriction enzymes with varying numbers of sites within endogenous xenotropic-related sequences. The digested DNA was then resolved on agarose gels. Southern blots of digested DNA were hybridized with the pXenv probe specific for the env sequence of xenotropic viral sequences. The number of hybridizing bands varied from 7 to 19, depending on the restriction enzyme and inbred background. Most mutant strains were identical in their restriction patterns to the respective background strains. However, two B6 mutant strains, KH84 and HZ54, differed from C57BL/6 at a single band which appeared to be inherited from BALB/c in the derivation of the two congenic strains. The HZ43 strain lacked a male-specific band shared by both C57BL/6 and BALB/c; this loss was evidently independent of the original mutation which was observed to be autosomal. However, the KH148B and KH84 strains on the C57BL/6 background lacked single B6 bands. Both mutants were classified as gain mutants. An examination of previous reciprocal graft rejection patterns and retrovirus linkage to non-H-2 H loci indicated a strong inverse relationship between a linked retroviral sequence and presentation of a non-H-2 H antigen. This inverse correlation is consistent with reports of gene inactivation following retroviral insertion.