ABSTRACT
Restriction endonuclease analysis (REA) typing using HindIII enzyme is a highly discriminatory, reproducible, and consistent method of genetic typing of Clostridium difficile (CD) isolates. REA typing analyzes CD whole cellular DNA on two levels of discrimination: REA Group designation and REA Type designation, which distinguishes specific subtypes within the REA Group. This methodology has enabled the tracking of epidemiologically significant CD strains over time and in some cases has allowed documentation of the evolution of previously rare REA Group strains that have subsequently become epidemic. The chapter details the methods used to isolate and purify CD colonies from stool samples, to obtain intact, full-length whole cellular DNA from CD isolates by use of guanidine-EDTA solution, and to analyze the HindIII-digested DNA after electrophoretic separation on agarose gels.
Subject(s)
Bacterial Typing Techniques , Clostridioides difficile/classification , DNA, Bacterial/genetics , Deoxyribonuclease HindIII/chemistry , Restriction Mapping/methods , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , DNA, Bacterial/isolation & purification , DNA, Circular/genetics , DNA, Circular/isolation & purification , Electrophoresis, Agar Gel/instrumentation , Electrophoresis, Agar Gel/methods , Enterocolitis, Pseudomembranous/diagnosis , Enterocolitis, Pseudomembranous/microbiology , Feces/microbiology , Humans , Prohibitins , Restriction Mapping/instrumentationABSTRACT
SUMMARY: The R/Bioconductor package HiTC facilitates the exploration of high-throughput 3C-based data. It allows users to import and export 'C' data, to transform, normalize, annotate and visualize interaction maps. The package operates within the Bioconductor framework and thus offers new opportunities for future development in this field. AVAILABILITY AND IMPLEMENTATION: The R package HiTC is available from the Bioconductor website. A detailed vignette provides additional documentation and help for using the package.
Subject(s)
Chromosome Mapping/methods , Restriction Mapping/methods , Software , Animals , Chromosome Mapping/instrumentation , Chromosomes, Human, Pair 14/chemistry , Chromosomes, Human, Pair 14/genetics , Data Display , Humans , Mice , Molecular Conformation , Restriction Mapping/instrumentationABSTRACT
A double-strand DNA (ds DNA) microarray was fabricated to analyze the structural perturbations caused by methylation and the different base mismatches in the interaction of the restriction endonucleases HpaII and MspI with DNA. First, a series of synthesized oligonucleotides were arrayed on the aldehyde-coated glass slides. Second, these oligonucleotides were hybridized with target sequences to obtain ds DNA microarray, which includes several types of double strands with the fully methylated, semi-methylated, and unmethylated canonical recognition sequences, semi-methylated and unmethylated base mismatches within the recognition sequences. The cleavage experiments were carried out under normal buffer conditions. The results indicated that MspI could partially cleave methylated and semi-methylated canonical recognition sequences. In contrast, HpaII could not cleave methylated and semi-methylated canonical recognition sequences. HpaII and MspI could both cleave the unmethylated canonical recognition sequence. However, HpaII could partially cleave the sequence containing one GG mismatch and not cleave other base mismatches in the corresponding recognition site. In contrast, MspI could not recognize the base mismatches within the recognition sequence. A good reproducibility was observed in several parallel experiments. The experiment indicates that the microarray technology has great potentials in high-throughput identifying important interactions between protein and DNA.
Subject(s)
Base Pair Mismatch , DNA-Cytosine Methylases/analysis , DNA-Cytosine Methylases/chemistry , Deoxyribonuclease HpaII/analysis , Deoxyribonuclease HpaII/chemistry , Oligonucleotide Array Sequence Analysis/methods , Oligonucleotides/analysis , Oligonucleotides/chemistry , Binding Sites , DNA Restriction Enzymes/analysis , DNA Restriction Enzymes/chemistry , DNA-Binding Proteins/analysis , DNA-Binding Proteins/chemistry , Equipment Failure Analysis , Oligonucleotide Array Sequence Analysis/instrumentation , Protein Binding , Reproducibility of Results , Restriction Mapping/instrumentation , Restriction Mapping/methods , Sensitivity and SpecificityABSTRACT
This study explores the restriction display-polymerase chain reaction (RD-PCR) application of a new chip-based nucleic acid analysis system (Agilent 2100 bioanalyser) in a gene differential expression study. Total RNAs is extracted from Saccharomyces cerevisiae, double-stranded complementary DNA (cDNA) is synthesised by reverse transcription from the purified messenger RNA (mRNA), RD-PCR conducted to obtain the cDNA fragments and bioanalyser and agarose gel electrophoresis compared for the analysis of RD-PCR products. The bioanalyser proved to be faster and more sensitive in separating and detecting gene fragments, and was also able to compare different gene fragments quantitatively. Using this technology, comparison of several differential gene fragments is performed.
Subject(s)
Gene Expression/genetics , Polymerase Chain Reaction/instrumentation , Saccharomyces cerevisiae/genetics , DNA/analysis , Electrophoresis, Agar Gel , Heat-Shock Proteins/genetics , Restriction Mapping/instrumentation , Saccharomyces cerevisiae/cytology , Sensitivity and SpecificityABSTRACT
Many new techniques in biomolecular chemistry and genomic analysis require the immobilization of molecular reagents on specially prepared surfaces. However, the process of molecular fixation often interferes with or precludes the use of standard in vitro biochemical assays. Optical mapping is an emergent technology for genomic analysis which relies on the biochemical activity of DNA fixed to silanized glass surfaces. Optical mapping surfaces have been shown to be compatible with restriction endonucleases and a variety of DNA polymerases. The essential properties of biochemically active surfaces are poorly understood in most of the current technologies which utilize molecular fixation, including optical mapping. The purpose of this study is to use the powerful technique of atomic force microscopy, in combination with informative enzymatic assays, to correlate biochemical activity with microscopic surface structure. The results presented provide meaningful insight into the effect of surface preparation on the biochemical accessibility of surface-bound molecules. Novel analysis which may facilitate the automation of optical mapping is presented.
