Biomedical subjects
Publications and source records attributed to E M Southern.
Clones of cDNAs have several practical advantages as start points for making physical maps of genomes: they are mainly single copy; they expand the map because of the introns in the genomic version of the gene; and they are conserved between species. They also address biologically important regions of the genome. Sequence information helps to identify new gene functions.
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In this paper we describe a modification to the lambda vector EMBL3 which greatly expedites the construction of restriction maps of cloned DNA sequences. In the modified vector, EMBL3cos, all the phage coding sequences are placed to the right of the cloning sites so that the left cohesive end is separated by only 200bp, rather than 20kb (as in conventional lambda vectors), from the inserted DNA fragment. We show that reliable restriction maps can be rapidly constructed from partial digests of clones made in this vector by labelling the left cohesive end with a complementary 32P-labelled oligonucleotide. In addition, we quantify the restriction of clones containing human DNA by the McrA and McrB systems of E. coli and show that the use of Mcr- plating strains can increase the yield of recombinant phage up to tenfold, to give cloning efficiencies of greater than or equal to 10(7) pfu/microgram of human DNA.
Linkage maps are limited by the number of recombinations that can be scored in human pedigrees to a resolution of ca. 1 centimorgan (relative distance between genes on a chromosome having a crossover value of 1%) which is estimated to be about 10 megabases. Molecular maps can be formed at any resolution down to the base sequence. To complement the linkage approach, the most useful molecular map would be one that helped to locate disease loci, by using restriction fragment length polymorphisms (RFLPS) and accurate localization of recombinations, and which then helped to find candidate genes in this region, by providing the positions of coding sequences. This paper discusses the appropriate form and scale of such a map, how it can be produced with methods now available, and the most efficient strategy for building the map, based on present knowledge of the organization of the human genome.
Detailed analysis of a large region of genomic DNA is facilitated by generating overlapping clones covering the entire region. These clones are usually obtained by bidirectional "walking" using either bacteriophage lambda or cosmid cloning vectors. This is a slow procedure when starting from a single start site. Multiple start sites are an advantage, and here we describe a method of generating clones from an extensive region of the Duchenne muscular dystrophy locus by preparative pulsed field gel electrophoresis using the chromosome of interest isolated in a cell hybrid. We have generated 12 clones mapping to an 840-kb SfiI fragment of DNA from the Xp2.1 region of the X chromosome, where the DMD gene has been localized. Further localization of these clones to the four subregions of the 840-kb fragment indicates that the clones are distributed throughout the fragment. The feasibility of using this approach to generate probes close to other loci is discussed.
The idea that large DNA molecules adopt a stretched conformation as they pass through gels suggests a simple mechanism for the separation of DNA by crossed field electrophoresis: at each change in field direction a DNA molecule takes off in the new direction of the field by a movement which is led by what was formerly its back end. The effect of this ratcheting motion is to subtract from the DNA molecule's forward movement, at each step, an amount which is proportional to its length. We find that this model explains most of the features of the separation, and we describe experiments, using a novel electrophoresis apparatus, which support the model. The apparatus turns the gel between two preset orientations in a uniform electric field at preset time intervals. This separation method has the practical advantage over some others that the DNA molecules follow straight tracks. A further advantage is that the parameters which determine the separation are readily predicted from the simple theory describing their motion.
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We have developed a large format digital scanner for several applications in nucleic acid analysis. Here we describe the scanning of autoradiographs of DNA sequencing gels and a set of programs for reading the base sequence. The programs correct distortions in the gel, recognize bands by their characteristic shape and assign bases to bands by weighting band position and intensity. The sequence read in this way is as accurate as that read by an expert.
UV-irradiation of DNA can inhibit the activity of certain restriction endonucleases because of thymine dimer formation within the enzyme recognition sequence. The number of sites affected depends upon the dose of UV, thus making it easier to control the extent of enzyme digestion than by either limiting the digestion time, or the amount of enzyme. Restriction-site maps of bacteriophage lambda recombinants are readily produced by labelling DNA using a radioactive oligonucleotide that is complementary to either the left or right cohesive end of lambda, irradiating the DNA with UV light, limit digesting with the appropriate enzyme, and calculating the size of the fragments detected after gel electrophoresis and autoradiography.
Pre-reassociation of human clone probes, containing dispersed highly repeated sequences, (e.g. Alu and KpnI families), with a large excess of sonicated total human DNA allows signal from single and low copy number components to be detected in transfer hybridisations. The signal from non-dispersed repeated sequences is reduced to single copy levels. The procedure, which is simple and quick, is illustrated using model combinations of well characterised cloned probes, and is applied to a sample of randomly chosen cosmid clones. A theoretical assessment is presented which may be useful to those wishing to use this procedure.
In this review evidence for the possible relationship between higher order chromosome structure and the distribution of tandem and dispersed repeated sequences in DNA has been examined. Evidence from studies of chromosome diminution in lower eukaryotes suggests that simple sequence DNAs may have a germ-line function and, in mammals, changes in simple sequence methylation, associated with changes in chromosome condensation, support the idea that the centromeric and telomeric heterochromatin may have a function in germ-line cells. For the major families of dispersed repeats, the weight of available evidence suggests that if they do play a role in chromosome organization, it is not an important one. Cytosine methylation is suggested as a candidate for a role in organizing the chromatin. Long-range patterns of methylation in vertebrates, the relationship of this to gene structure, and the association of changes in methylation with gene activity accord with current evidence linking specific nuclear structures to defined points in coding regions.
The electrophoretic mobilities of DNA polymer fragments in an agarose gel have been measured from a photograph of the gel by different methods and converted to lengths by the reciprocal method. The method of measurement can introduce large errors in the length estimates. The use of a digital microdensitometer to obtain optical density profiles of gel tracks with subsequent computer processing to find peak positions was found to give the most accurate DNA lengths.
Relationships between DNA length and electrophoretic mobility in an agarose gel have been compared by estimating the lengths of known DNA polymer fragments, using other fragments in the series as standards. Global estimates were made using 10 fragments as standards; local estimates were made using the two closest fragments on either side of the unknown as standards. Most relationships were fitted by least squares. All the relationships gave more accurate local than global estimates. The most accurate results were obtained using the reciprocal relationship, where the maximum error in the local estimates was less than 0.1%. The semilog relationship gave the least accurate results, with a maximum error in the local estimates of almost 5%. The polymer fragments were also used as standards to estimate the lengths of lambda DNA restriction fragments. Here the estimates were in error by up to 3%, indicating the influence of base composition and sequence on electrophoretic mobility.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.