DNA sequence similarity recognition by hybridization to short oligomers

Methods are disclosed for the comparison of nucleic acid sequences. Data is generated by hybridizing sets of oligomers with target nucleic acids. The data thus generated is manipulated simultaneously with respect to both (i) matching between oligomers and (ii) matching between oligomers and putative reference sequences available in databases. Using data compression methods to manipulate this mutual information, sequences for the target can be constructed

Global expression mapping of mammalian genomes

he aim of genome projects is to decipher all the information contained within the DNA of an organism and to study the way this information is processed in physiological processes. It is believed that more than 95% of the information content of the mammalian genome is represented in the protein coding sequences that make up only approximately 2% of the DNA sequence. Consequently much effort is being invested in the study of coding sequences in the form of cDNA analysis. This thesis is concerned with the development of a new strategy for a highly parallel approach to analyse entire cDNA libraries. The strategy is based upon generating sufficient sequence information to identify uniquely more than 100,000 cDNA clones by hybridisation with short oligonucleotides, typically 7 - 10 mers. Each oligonucleotide is hybridised to all cDNA clones in parallel and under stringent conditions positively identifies a subset (3 - 10%) of clones. Oligonucleotides are designed in such a way that each will positively identify a different subset of clones and statistical simulations estimate that approximately 200 such hybridisation events are required to identify uniquely upto 100,000 cDNA sequences. Such a fingerprint can be generated from many cDNA libraries constructed from different tissue mRNAs and will not only lead to the identification of most sequecnes expressed from the genome but also indicate the level of expression by determining the number of times any given sequence is represented across different cDNA libraries. A human foetal brain cDNA library has been constructed and 100,000 clones arrayed into microtitre plates and on nylon membranes. All the required technological developments have been carried out successfully and are presented. In excess of 200 oligonucleotide hybridisations have been performed on a subset of 32,000 cDNA clones and 1,000 sequenced control clones. A detailed analysis of the data on the control clones is presented and the implications for cDNA fingerprinting discussed

Introduction on Using the FastPCR Software and the Related Java Web Tools for PCR and Oligonucleotide Assembly and Analysis

This chapter introduces the FastPCR software as an integrated tool environment for PCR primer and probe design, which predicts properties of oligonucleotides based on experimental studies of the PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations. These include the standard PCR as well as the multiplex, long-distance, inverse, real-time, group-specific, unique, overlap extension PCR for multi-fragments assembling cloning and loop-mediated isothermal amplification (LAMP). It also contains a built-in program to design oligonucleotide sets both for long sequence assembly by ligase chain reaction and for design of amplicons that tile across a region(s) of interest. The software calculates the melting temperature for the standard and degenerate oligonucleotides including locked nucleic acid (LNA) and other modifications. It also provides analyses for a set of primers with the prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity as well as a primer dilution and resuspension calculator. The program consists of various bioinformatical tools for analysis of sequences with the GC or AT skew, CG% and GA% content, and the purine–pyrimidine skew. It also analyzes the linguistic sequence complexity and performs generation of random DNA sequence as well as restriction endonucleases analysis. The program allows to find or create restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It performs efficient and complete detection of various repeat types with visual display. The FastPCR software allows the sequence file batch processing that is essential for automation. The program is available for download at http://primerdigital.com/fastpcr.html, and its online version is located at http://primerdigital.com/tools/pcr.html.Peer reviewe

Fingerprinting and characterisation of Escherichia coli isolates using DNA arrays

Two commercially available DNA whole genome Escherichia coli K12 arrays were compared to identify a subset of markers for typing. The arrays were identical in probe composition but different in substrate (membrane and glass slide arrays) and probe preparation (radio- and fluorescent-labelled). Labelled genomic E. coli DNA from five strains of the E. coli reference (ECOR) collection (ATCT35320 - ATCX35324) and E. coli K12 were hybridised against these arrays. A group of 1240 putative markers was identified on the membrane arrays and 649 were found on the glass slide arrays. Only a small proportion of these sequences (8%) was found through both platforms. Variability in the hybridisation signals from duplicate experiments made it difficult to identify useful markers. In order to investigate whether this technology could be used for characterising or typing E. coli strains, an array for the detection of 29 pathogenicity markers in E. coli strains was produced. This array was used with eight reference strains, including different pathotypes, 72 strains from the ECOR collection, and 49 clinical isolates. A wide range of E. coli pathogenicity markers was detected. The pathogenicity markers that were most common include chuA and iucC, which are both involved in iron metabolism. Additionally, the clinical isolates were grouped into clusters different from groupings based on biochemical tests. This demonstrates that the use of pathogenicity array typing can complement diagnostic tests on clinical E. coli isolates. An extended, second-generation, pathogenicity marker array containing 75 probes was made. The extended array successfully distinguished between ten closely related isolates from an outbreak of urinary tract infections, while previous tests were unable to do so. This array has the potential for providing a rapid and novel means of characterising pathogenic isolates

Polymerase Chain Reaction

Polymerase chain reaction (PCR) is an efficient and one of the most common methods used in biological sciences for in vitro multiplication of a target DNA molecule. The technique has significantly contributed in changing and developing different fields of biological sciences since 1980s. PCR has a vital role in supporting the processes involved in genetic engineering, particularly the cloning of DNA fragments used to modify the genomes of microorganisms, animals, and plants. Consequently, the technique has numerous applications in fundamental and applied research in medicine agriculture, environment, and bio-industry. The main focus of this chapter is to describe briefly the principles, methodology, various types, and applications of PCR in different fields. Besides, different components of PCR, trouble shooting during the execution, and limitations of the techniques are also outlined

Evaluation and Optimization of Bioinformatic Tools for the Detection of Human Foodborne Pathogens in Complex Metagenomic Datasets

Foodborne human pathogens pose a significant risk to human health as each year one in six Americans becomes sick from one of over 31 known human foodborne pathogens. Due to the differences in their growth requirements, current detection assays can only detect one to a few of these pathogens per single assay. Metagenomics, an emerging field, allows for an entire community of organisms to be analyzed from DNA or RNA sequence data generated from a single sample, and therefore has the potential to detect any and all foodborne pathogens present in a single complex matrix. However, currently available bioinformatic pipelines for metagenomic sequence analysis require extensive time and high computer power inputs, often with unreliable results. The objectives of this study are 1) to evaluate community profiling bioinformatic pipelines, mapping pipelines and a novel pipeline created at Oklahoma State University, E-probe Diagnostic Nucleic-acid Analysis (EDNA), for the detection of S. enterica (as a model foodborne pathogen) in metagenomic data, 2) to optimize EDNA pipeline for sensitive detection of the S. enterica in metagenomic data, and 3) to simultaneously detect multiple foodborne pathogens from a single metagenomic sample. EDNA was able to detect S. enterica in metagenomic data in approximately five minutes compared to the other pipelines, which took between 2-500 hours. The optimized parameters for the EDNA pipeline were limited to using cleaned Illumina data with a read depth of one. The minimum BLAST E-value was set to 10^-3 for curation. For detection the minimum percent identity was set to 95% and the minimum query coverage to 90% with an E-probe length of 80 nt. These new parameters significantly improved the sensitivity of the assay 100-fold, from 10^3 S. enterica cells detected by the original EDNA pipeline to just 10 cells. In the simultaneous detection of multiple foodborne pathogens, EDNA detected three additional pathogens Listeria monocytogenes, Campylobacter jejuni and Shiga toxin producing Escherichia coli at ten contamination levels in less than ten minutes and provided new detection insights into read abundance as it corresponds to pathogen cell numbers

Biodegradation of Synthetic Polymers in the Aquatic Environment

Biodegradation of synthetic polymers can be a sophisticated property for intelligent and sustainable products that offer complex benefits for specific applications. There are many entry paths for synthetic polymers that can accumulate in the aqueous and especially marine environment and little is known about their biodegradation especially in the aquatic environment. The difficulties with determining biodegradation in those environments are based on the absence of appropriate methods and also the fact that these environments often prove low biodegradation rates. It is also complicated to detect biodegradation on polymeric substances because of the high molecular weight, water insolubility and difficult molecular structure making it hard to detect biodegradation products. This work provides an overview of the actual status of research and investigates on different methods of biodegradation tests in the aquatic environment with selected synthetic polymers

Biodegradation of Synthetic Polymers in the Aquatic Environment

Biodegradation of synthetic polymers can be a sophisticated property for intelligent and sustainable products that offer complex benefits for specific applications. There are many entry paths for synthetic polymers that can accumulate in the aqueous and especially marine environment and little is known about their biodegradation especially in the aquatic environment. The difficulties with determining biodegradation in those environments are based on the absence of appropriate methods and also the fact that these environments often prove low biodegradation rates. It is also complicated to detect biodegradation on polymeric substances because of the high molecular weight, water insolubility and difficult molecular structure making it hard to detect biodegradation products. This work provides an overview of the actual status of research and investigates on different methods of biodegradation tests in the aquatic environment with selected synthetic polymers