Biomedical subjects
Publications and source records attributed to W Bains.
This article reports the preliminary results of a search for patterns in the strand asymmetry and local self-similarity in mammalian nuclear DNA. The study provides the basis for statistical investigations of larger-scale DNA structure, which may be correlated with genetic mechanisms or with gene function.
DNA manipulation techniques have allowed the isolation and identification of many genes which cause disease in man. The same techniques are now being automated, and are on the threshold of application to more common and more complex genetic predispositions to diseases such as diabetes and schizophrenia. This article outlines the technology behind this development, and discusses some of the social, legal and ethical difficulties that widespread screening for genetic traits will raise. It is argued that these issues must be considered now, so that considered social responses to this technology are in place in the five years it is likely to take to develop. The techniques of DNA manipulation that have led to recombinant proteins and biopharmaceuticals have also led to an unprecedented increase in understanding of human genetics, and a corresponding increase in the practical side of human genetics--medical genetic diagnosis. As a result, medical genetics is moving from a specialty of statisticians and cytologists into the mainstream of medicine, and will soon be the concern of every hospital, and probably of every GP. This offers huge benefits to patients, but also threatens misunderstanding and even the potential of abuse if this sophisticated understanding of our fundamental natures is used without precision.
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Hybridization of a large panel of oligonucleotides to a target DNA could be used to determine the sequence of that target. The inherent ambiguity of the reconstruction limits the length of DNA that can be sequenced by such an approach. Here I show that constraining the reconstruction by assuming that the target DNA contains an open reading frame allows longer DNAs to be sequenced. In the case of ideal hybridization using mixed 11-mer probes, the amount of DNA that can be sequenced is extended from 700 bases to 2400 bases. This is sufficient to sequence substantial portions of most cDNA clones, and suggests that oligonucleotide hybridization could be a practical method of generating large amounts of sequence data from cDNA libraries.
The rate at which bases are replaced in mammalian DNA is influenced by the sequence involved. I have modified a multiple sequence alignment and comparison program to take account of the observed patterns of base replacement in mammals, and used the program to analyse 100 human Alu sequences. The results show that using a sequence matching matrix based on the actual rate of base replacement, as opposed to arbitrary base matching matrix, gives significant differences in the way that sequences are aligned and compared with each other. Use of the observed matching matrix gives significantly better discrimination between sub-families of Alu sequences than conventional methods, and shows that comparison systems which take account of the mechanism of mutation give more biologically realistic results.
The codons for some conserved amino acids are found to be the same between homologous genes from different species when the statistics of codon usage would suggest that they should be different. I examine whether this 'coincidence' of codon usage could be due to genetic mechanisms homogenising the DNA around specific sites. This paper describes the further analysis of the coincident codons in 19 genes (a total of 96 homologues) for slippage. Coincident codons arise in contexts of increased sequence simplicity, and have a high chance of occurring within sequences similar to the recombination-prone minisatellite 'core' sequence. This suggests a role of genetic homogenisation in their generation.
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I have analysed the local sequence context of base replacement changes in 78 processed pseudogenes. Transversions occur more often than transitions in a ratio of 3.37 to 1, and G:C is replaced 1.4 times more frequently than A:T. In addition, the bases to the 5' and 3' of the mutating base also influence the rate at which bases change, purine:pyrimidine and pyrimidine:purine pairs changing 1.2 times as fast as purine:purine and pyrimidine:pyrimidine pairs. I discuss implications of this for the mechanism of DNA polymerization in mammals.
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I have conducted a general analysis of the practicability of using oligonucleotide hybridization to sequence DNA. Any DNA sequence may be sequenced by hybridization with a complete panel of oligonucleotides. However, sequencing DNA segments over 2 kb long requires an unrealistic number of hybridization reactions. The optimal protocol is to hybridize 7-mer or 8-mer mixed oligonucleotide probes to immobilized DNA fragments 80 bp long: should this prove impractical, hybridization of labeled 270-bp fragments to immobilized mixed 10-mers is a potential alternative. Both protocols require no more experiments to sequence large regions of DNA than conventional m13-based sequencing and are much easier to automate, thus reducing the requirements for skilled personnel. In the ideal case, hybridization sequencing reduces the number of experiments required to sequence megabase DNA by 90%.
The analysis of a genomic loci containing human histone H3.3 processed pseudogenes, has revealed two regions that are unusually rich in other retroposons. At one of the loci the H3.3 pseudogene is itself interrupted by 2 Alu repetitive sequences. The characterization of these two recently transposed Alus provides confirmation of the "multiple origin" hypothesis of these repetitive elements. The unusual occurrence of 3 different types of retroposons in a small region suggests that there may be particular chromosomal regions that are hot spots for retroposon insertion.
The approach I describe to using DNA probes in diagnostic tests is simpler than most existing formats. DNA in a sample is labeled by chemical reaction with bisulfite and methylamine to generate a sulfonated derivative. The DNA need not be purified to do this. The labeled sample is then incubated with an unlabeled, purified probe DNA, which is immobilized to a solid support. The amount of label remaining on the solid support after washing is detected by a monoclonal antibody that recognizes modified cytosines. The intensity of the signal depends on the amount of target DNA in the sample. Detection limits depend on the amount of immobilized DNA and on the degree of physical entrapment of the labeled DNA in sample material, but can be as low as 5 pg. This format is well suited to automation for use with existing robotic enzyme immunoassay procedures.
We have used Fragmentation Sequencing logic to analyse the repetition structure of several large human genomic genes. The method, based on a proposed laboratory scheme for DNA sequencing, detects short sequences which are repeated near, but not necessarily adjacent, to each other (cryptically simple DNA). We find a low frequency of such repeats. There is a slight excess of such repeats in introns over exons, and a slight but significant excess in genomic DNA over random DNA, confirming that cryptically simple sequences are over-represented in the genome. The analysis suggests that Fragmentation Sequencing will be a suitable method for sequencing large mammalian genes.
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