Kinetics of target site localization of a protein on DNA: a stochastic approach

It is widely recognized that the cleaving rate of a restriction enzyme on target DNA sequences is several orders of magnitude faster than the maximal one calculated from the diffusion--limited theory. It was therefore commonly assumed that the target site interaction of a restriction enzyme with DNA has to occur via two steps: one--dimensional diffusion along a DNA segment, and long--range jumps coming from association/dissociation events. We propose here a stochastic model for this reaction which comprises a series of 1D diffusions of a restriction enzyme on non-specific DNA sequences interrupted by 3D excursions in the solution until the target sequence is reached. This model provides an optimal finding strategy which explains the fast association rate. Modeling the excursions by uncorrelated random jumps, we recover the expression of the mean time required for target site association to occur given by Berg & al. \cite{berg81}, and we explicitly give several physical quantities describing the stochastic pathway of the enzyme. For competitive target sites we calculate two quantities: processivity and preference. By comparing these theoretical expressions to recent experimental data obtained for \textit{Eco}RV--DNA interaction, we quantify: i) the mean residence time per binding event of \textit{Eco}RV on DNA for a representative 1D diffusion coefficient, ii) the average lengths of DNA scanned during the 1D diffusion (during one binding event and during the overall process), iii) the mean time and the mean number of visits needed to go from one target site to the other. Further, we evaluate the dynamics of DNA cleavage with regard to the probability for the restriction enzyme to perform another 1D diffusion on the same DNA substrate following a 3D excursion.Comment: 10 pages, 8 figure

Computer Aided Simulation of DNA Fingerprint Amplified Fragment Length Polymophism (AFLP) Using Suffix Tree Indexing and Data Mining

AFLP is one of the DNA Fingerprinting techniques which have broad application as genetic marker in various fields. Begin with the DNA sequence digestion using one or more particular restriction enzyme, ligation of the adapters to the overhanging sticky ends followed by DNA fragments amplification using PCR. The PCR reaction uses primers that match the adapter sequence and have some (1 to 3) dditional “selective” bases which could be any bases, this reduces the number of bands that will be amplified. Such technique intended to increase the amplified fragments peculiarity so the polymorphism of the organism being studied could be well visualized by gel electrophoresis. The computer aided of AFLP simulation developed in this research was aimed to predict this electrophoresis result by simulate the digestion, ligation and PCR process using some pattern recognition algorithm applied to the DNA sequence from online databases. Through this simulation the researcher could determine the best combination of restriction enzyme and selective bases for their laboratory experiment. Suffix tree indexing was conducted during the exploration process of the genome sequence (in FASTA format) to find the restriction sites rapidly and create fragments of it. Data modeling enable the system draws the fragments into virtual DNA’s electrophoresis pattern. Data mining accomplish the simulation by exploring overall possible virtual DNA’s electrophoresis pattern and determine the best restriction enzyme and selective bases combination by calculating certain quantitative criteria

PCR-RFLP Using BseDI Enzyme for Pork Authentication in Sausage and Nugget Products

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using BseDI restriction enzyme had been applied for identifying the presence of pork in processed meat (beef sausage and chicken nugget) including before and after frying. Pork sample in various levels (1%, 3%, 5%, 10%, and 25 %) was prepared in a mixture with beef and chicken meats and processed for sausage and nugget. The primers CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b (cyt b) gene and PCR successfully amplified fragments of 359 bp. To distinguish existence of porcine species, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed pig mitochondrial DNA was cut into 131 and 228 bp fragments. The PCR-RFLP species identification assay yielded excellent results for identification of porcine species. It is a potentially reliable technique for pork detection in animal food processed products for Halal authentication

DNA Mapping Algorithms: Strategies for Single Restriction Enzyme and Multiple Restriction Enzyme Mapping

An approach to high-resolution restriction-fragment DNA mapping, known as Multiple-Restriction-Enzyme mapping (MRE mapping), is present. This approach significantly reduces the uncertainty of clone placement by using clone ends to synchronize the position in of clones within different maps, each map being constructed from fragment-length data produced by digestion of each clone with a specific restriction enzyme. Maps containing both fragments-length data and clone-end data are maintained for each restriction enzyme, and synchronization between two such maps is achieved by requiring them to have compatible clone-end map projections. Basic definitions of different kinds of maps, such as restriction sites maps, restriction fragment maps and clone end maps, are presented. Several specifications notations, such as sequence-set notation and sequence-set-tree notation, for describing the structure of these maps, are defined. Basic concepts, such as the match/merge approach to map incorporation, extension vs. assimilation and ambiguity, are exposed. Supporting techniques, such as window sizing, window placement, and ambiguity resolution, are also discussed. A mathematical analysis of how MRE mapping effects false positives and false negatives is presented. For concreteness, MRE mapping is presented using a specific methodological framework. However, many of the concepts and techniques have a wider range of use than just high-resolution restriction-fragment mapping

The Efficiency of Transgenesis by Restriction Enzyme Mediated Integration - Sperm Mediated Gene Transfer (REMI-SMGT)

The present study tends to  test the validity of REMI-SMGT and to evaluate the efficiency of REMI-SMGT on generating other mammalian species rather than the sheep that made only by one group and to observe the possibility of doing so by using rabbits as a model for this approach and also to reduce the cost of REMI-SMGT by substituting liposomes and highly cost effective media with a high efficient, non-cost effective substitute. Direct protective relationship of liposome with DNA and seminal fluid was identified compared with DMSO.  While different treatments (linearized DNA – restriction enzyme – liposome complex, DNA – restriction enzyme – DMSO complex, DNA –DMSO complex, DNA –liposome complex, and even naked DNA) were all found to be successful to internalize inside the head of the sperm according to PCR results, only three (one by restriction enzyme – liposome treatment and two by restriction enzyme – DMSO treatment) out of fourteen new born babies were found to be transgenic by PCR. Despite the absolute ability of exogenous DNA to be internalized inside rabbit’s sperm head only few percent of transgenic babies were obtained. This may not reflect the weakness of restriction enzyme mediated transgenesis technique itself but it reflects the inability of recombinant sperm to fertilize superovulated oocyte compared with their normal counterparts. Comparable results were found between liposome and DMSO treatment which may reflect direct relationship of DMSO with the cell membrane instead of with the exogenous DNA itself as what is found with liposome. Key words : REMI-SMGT , PCR , DNA – restriction enzyme , DMS

Lack of class I H-2 antigens in cells transformed by radiation leukemia virus is associated with methylation and rearrangement of H-2 DNA

Transformation of murine thymocytes by radiation leukemia virus is associated with reduced expression of the class I antigens encoded in the major histocompatibility complex (MHC) and increased methylation and altered restriction enzyme patterns of MHC DNA. These changes may play a role in host susceptibility to virus-induced leukemogenesis and accord with the notion that viral genomes play a regulatory function when they integrate adjacent to histocompatibiity genes

The effect of DNA structure and restriction enzymes on transformation efficiencies in Neurospora crassa

Addition of the appropriate restriction enzyme to linearized transforming DNA has been shown to increase transformation efficiencies in organisms as diverse as Saccharomyces cerevisiae (Schiestl and Petes 1991 Proc. Nat. Acad. Sci. USA 88:7585-7589) and Dictyostelium discoideum (Kuspa and Loomis 1992 Proc. Nat. Acad. Sci. USA 89:8803- 8807). This process has been described as REMI, for restriction enzyme-mediated integration. We have examined the effect of restriction enzyme addition on transformation efficiencies in Neurospora crassa. The frequency of cotransformation of a qa-2 inl double mutant with two plasmids [one containing the selectable marker qa-2+ (quinate utilization) and the other containing inl+ (inositol)] was also examined, as was the generation of stable versus abortive transformants

Using CRISPR-Cas9 as a Restriction Enzyme

Restriction digests are a commonly utilized process for cleaving DNA at specific, but relatively common sites. Restriction enzymes have widespread use in DNA manipulation. CRISPR/Cas9 is a recently identified endonuclease which utilizes a customizable guide sequence to recognize and cut specific ~20 bp sites located in a DNA sequence. This preliminary research aimed to exploit the potential benefit of DNA restriction using the CRISPR/Cas9 procedure through alterations of different components involved in that system. We sought to refine existing CRISPR/Cas9 protocols and make a budget friendly, user-selectable CRISPR/Cas9 restriction digest protocol. The motivation for this research was to simplify and adapt known CRISPR protocols in hopes of using CRISPR as a targeted restriction enzyme. This project yielded negative results, however, important insights into the dilution of target sequences was achieved