Biomedical subjects
Publications and source records attributed to D K Green.
Telomeric DNA in the skin cells of 21 human subjects aged between 0 and 92 years was quantified by determining the length of the telomeric smear and the relative amount of TTAGGG repeat sequences. Both telomere length and quantity of telomeric repeat sequences were found to decrease significantly with age. Telomere loss has previously been postulated to be a caused of cell senescence.
The design and construction of a transistor-driven hexagonal contour-clamped homogeneous electric field (CHEF) apparatus is discussed in detail. The addition of computer control of pulsed-field timings and experiment duration gives rise to an efficient electrophoresis tool designed to achieve separation of DNA molecules in different size groupings. In particular, pulse time regimes which lead to the monotonic separation of DNA molecules ranging from 90 kbp to over a megabase pair are demonstrated. Theoretical treatment of electric field clamping with transistor-driven multiple electrodes is supported by measurements and by the actual performance of electrophoretic separation of yeast chromosomes. The large sample capacity of gels run in this apparatus coupled with the modest power requirements necessary to provide a homogeneous electric field offer significant advantages over earlier CHEF designs.
Analysing and sorting human chromosomes by flow cytometry is a powerful tool in the hands of the molecular biologist. Because of the large number of chromosomes analysed by flow, typically 10(5) for each sample, estimates of the distribution of DNA throughout the chromosome complement of an individual can be made to within three megabase pairs. Rearrangements of DNA in this size range can often be clearly seen and measured by flow cytometry in situations where it was not obvious by traditional cytogenetics. The production of enriched samples of a particular chromosome by flow sorting and the subsequent construction of DNA libraries has played an important part in mapping genes to particular hereditary diseases. Techniques now exist which allow the hybridization of a few thousand sorted chromosomes with characterized or uncharacterized DNA probes to give relatively quick answers about specific chromosome genes. The process of obtaining a sorted chromosome sample from a growing population of peripheral blood lymphocytes or lymphoblastoid cells is compared with other methods of achieving similar results in molecular biological terms. Advances in flow cytometric techniques, which include slit-scanning and hybridization of DNA probes in suspension, are likely to improve the enrichment quality of specific sorted human chromosomes.
It is demonstrated that there are a number of advantages in using a mechanised microscope for scoring a large number of metaphase cells from human blood lymphocyte preparations. Following the development of an automatic metaphase spread finding machine based upon a large motorised microscope and a synchronous closed circuit television camera and flashing light source, a much smaller machine which is more appropriate to the cytogenetics laboratory, but with a similar metaphase finding performance has been constructed. The new machine which consists of a Cambridge Instruments 1 micron stepping microscope stage, a linear diode array scanner and a computer is described in detail. Metaphase finding performance figures for various orcein stained human blood lymphocyte preparations are given.
An automatic focusing technique for a computer-controlled optical microscope is described, which is based on an algorithm originally proposed by Mendelsohn and Mayall. The procedure is capable of attaining a focusing accuracy of less than 1/20 micron in the slide-objective distance. The dependence of focusing accuracy and time to focus on the scanner signal-to-noise ratio, and the change in the focus position with time, are discussed.
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