Automated film reader for DNA sequencing based on homomorphic deconvolution

Journal ArticleAn automated reader for electrophoresis based DNA sequencing methods is described that provides fast and accurate sequence determination. Digitized sequencing lanes are processed with homomorphic blind deconvolution in preparation for peak detection, interlane alignment, peak refinement and base calling. Initial reads from direct blot sequencing films have error rates of about 1% at the rate of 5 nucleotides/s. Typical read lengths are 500-600 nucleotides. The described reader is a significant improvement over existing readers and could be an essential component in the sequencing efforts of the Human Genome Project

Automated band mapping in electrophoretic gel images using background information

Some popular methods for polymorphism and mutation discovery involve ascertainment of novel bands by the examination of electrophoretic gel images. Although existing strategies for mapping bands work well for specific applications, such as DNA sequencing, these strategies are not well suited for novel band detection. Here, we describe a general strategy for band mapping that uses background banding patterns to facilitate lane calling and size calibration. We have implemented this strategy in GelBuddy, a user-friendly Java-based program for PC and Macintosh computers, which includes several utilities to assist discovery of mutations and polymorphisms. We demonstrate the use of GelBuddy in applications based on single-base mismatch cleavage of heteroduplexed PCR products. Use of software designed to facilitate novel band detection can significantly shorten the time needed for image analysis and data entry in a high-throughput setting. Furthermore, the interactive strategy implemented in GelBuddy has been successfully applied to DNA fingerprinting applications, such as AFLP. GelBuddy promises to make electrophoretic gel analysis a viable alternative to DNA resequencing for discovery of mutations and polymorphisms

Sequencing as a way of work : a history of its emergence and mechanisation - from proteins to DNA, 1945-2000

Imperial Users onl

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

A study of telomere and telomerase biology in the dog and cat

The primary aims of this project were to carry out a comprehensive investigation of telomere and telomerase biology in the dog and cat, and more specifically to investigate the potential of telomeres and telomerase as targets for novel cancer chemotherapeutics. The first experiments carried out were concerned with investigating mean telomere lengths in a wide age and tissue range of both the dog and cat. The protocol used for telomere length assessment was based on a DNA probe, Southern Blot and chemiluminescent technique referred to as Terminal Restriction Fragment analysis. Telomere lengths in peripheral blood samples taken from 112 dogs and 30 cats were found to range from 4.7 to 20.6 kb, and 9.6 to 23.5 kb respectively. These are similar to telomere lengths typically found in human samples (5-15 kb). The telomere lengths in a panel of normal canine and feline organ samples, tumour samples, and primary fibroblast cultures also did not differ significantly from these values. Telomere lengths decreased significantly with increased age in both species, and whilst gender did not have a significant effect in either species, an intriguing finding was that breed of pedigree dog had a significant effect on telomere length. Primary canine and feline fibroblasts were found to cease replicating and assume a senescent phenotype in vitro after an average of 10 and 16 population doublings respectively. Over the course of these population doublings, telomere attrition was shown to occur in both canine and feline cells, and averaged 175 and 130 bp/cell division respectively. In summary, telomeres in the dog and cat are of a similar size to that found in humans, and telomeric attrition has been shown to occur in both species in vivo and in vitro. Furthermore, loss of telomeric sequence is associated with the triggering of a senescent phenotype in both canine and feline fibroblasts in vitro. Telomerase activity studies used a commercially available assay, referred to as the Telomeric Repeat Amplification Protocol (TRAP). Telomerase activity was strongly down regulated in a wide range of somatic tissues of the dog and cat. Conversely, telomerase activity was detected in all canine and feline tumours assayed (19/19), and was also present in a panel of immortalised canine cell lines. These data linked telomerase with immortalisation and malignancy in the dog and cat, and have identified telomerase as a potential target for novel cancer ehemotherapeutics in companion animals. A pilot study to assess the efficacy of a reverse transcriptase inhibitor (azidothymidine triphosphate) as a telomerase inhibitor in two canine telomerase dependant cell lines produced inconsistent inhibition of telomerase and no discernable effect on telomere lengths. However, future use of this drug in combination with agents that utilize different modalities for targeting telomerase may produce more favourable results. (Abstract shortened by ProQuest.)