Search PubMed⌕ Search

Biomedical subjects

E M Southern

Publications and source records attributed to E M Southern.

At least 37 records · Page 2Linked to original sources
PubMed1997

Effects of base mismatches on joining of short oligodeoxynucleotides by DNA ligases.

The requirement for Watson-Crick base pairing surrounding a nick in duplex DNA to be sealed by DNA ligase is the basis for oligonucleotide ligation assays that distinguish single base mutations in DNA targets. Experiments in a model system demonstrate that the minimum length of oligonucleotide that can be joined differs for different ligases. Thermus thermophilus (Tth) DNA ligase is unable to join any oligonucleotide of length six or less, while T4 DNA ligase and T7 DNA ligase are both able to join hexamers. The rate of oligonucleotide ligation by Tth DNA ligase increases between heptamer and nonamer. Mismatches which cause the duplex to be shortened by fraying, at the end distal to the join, slow the ligation reaction. In the case of Tth DNA ligase, mismatches at the seventh and eighth position 5'to the nick completely inhibit the ligation of octamers. The results are relevant to mechanisms of ligation.

Bacteriophage T7↗
PubMed1997

Steric factors influencing hybridisation of nucleic acids to oligonucleotide arrays.

We have investigated the use of spacer molecules to reduce steric interference of the support on the hybridisation behaviour of immobilised oligonucleotides. These spacers are built up from a variety of monomeric units, using phosphoramidite chemistry, by condensation onto an amine-functionalised polypropylene support. The optimal spacer length was determined to be at least 40 atoms in length, giving up to 150-fold increase in the yield of hybridisation. The effects of different charged groups in the spacer were also examined, and it was shown that both positively and negatively charged groups in the spacer diminish the yield of hybridisation. Steric hindrance in hybridisation can also be a problem if the oligonucleotides attached to the support are too close to each other. Surface coverage was varied using a combination of cleavable and stable linkers, giving the highest hybridisation yields for surfaces containing approximately 50% of the maximum concentration of oligonucleotides.

Base Sequence↗
PubMed1997

Discovering antisense reagents by hybridization of RNA to oligonucleotide arrays.

Antisense reagents have the potential to modify gene expression by interacting with DNA or mRNA to down-regulate transcription or translation. There have been a number of successful demonstrations of antisense activity in vivo. However, a number of problems must be solved before the method's full potential can be realized. One problem is the need for the antisense agent to form a duplex with the target molecule. We have found that most regions of mRNAs are not open to duplex formation with oligonucleotides because the bases needed for Watson-Crick base pairing are involved in intramolecular pairing. Using arrays of oligonucleotides that are complementary to extensive regions of the mRNA target, we are able to find those antisense oligonucleotides which bind optimally. There is good correspondence between the ability of an oligonucleotide to bind to its target and its activity as an antisense agent in in vivo and in vitro tests. To understand more fully the rules governing the process of duplex formation between a native RNA and complementary oligonucleotides, we have studied the interactions between tRNAphe and a complete set of complementary dodecanucleotides. Only four of the set of 65 oligonucleotides interact strongly. The four corresponding regions in the tRNA share structural features. However, other regions with similar features do not form a duplex. It is clear that ab initio prediction of patterns of interaction require much greater knowledge of the process of duplex formation than is presently available.

Animals↗
PubMed1997

Selecting effective antisense reagents on combinatorial oligonucleotide arrays.

An array of 1,938 oligodeoxynucleotides (ONs) ranging in length from monomers to 17-mers was fabricated on the surface of a glass plate and used to measure the potential of oligonucleotide for heteroduplex formation with rabbit beta-globin mRNA. The oligonucleotides were complementary to the first 122 bases of mRNA comprising the 5' UTR and bases 1 to 69 of the first exon. Surprisingly few oligonucleotides gave significant heteroduplex yield. Antisense activity, measured in a RNase H assay and by in vitro translation, correlated well with yield of heteroduplex on the array. These results help to explain the variable success that is commonly experienced in the choice of antisense oligonucleotides. For the optimal ON, the concentration required to inhibit translation by 50% was found to be five times less than for any other ON. We find no obvious features in the mRNA sequence or the predicted secondary structure that can explain the variation in heteroduplex yield. However, the arrays provide a simple empirical method of selecting effective antisense oligonucleotides for any RNA target of known sequence.

Animals↗
PubMed1996

DNA chips: analysing sequence by hybridization to oligonucleotides on a large scale.

Complete sequences of genomes and comprehensive sets of cDNA sequences open the way to a hugh range of biological problems. There is a need for analytical methods that can deal with the large number of sequences in the data banks and, ideally, we would like to analyse all sequences together. Gel-based sequencing is a serial process that analyses one sequence at a time. Oligonucleotide arrays, or 'DNA chips', are miniature, parallel analytical devices, which could bring to sequence analysis and molecular genetics many of the advantages that semiconductor devices brought to computing.

Base Sequence↗
PubMed1996

High-density gridding: techniques and applications.

Much progress has been made in the development of techniques for constructing dense grids either of ligands, such as peptides and oligonucleotides, or of cloned nucleic acids. Such arrays are finding practical applications in the analysis of sequence variation and gene expression. Methods for carrying out large numbers of analyses in parallel will be essential for the genetic programme that is developing from large-scale sequencing projects.

Animals↗
PubMed1995

DNA fingerprinting by hybridisation to oligonucleotide arrays.

Most methods for analysing variation in DNA sequences rely on gel electrophoresis, a method which is difficult to automate and to quantify. Hybridisation to oligonucleotides bound to a solid surface offers alternatives, with the advantage that they can be more readily automated and lend themselves to powerful statistical analytical procedures. In this paper, we discuss a number of different ways in which oligonucleotide arrays could be used for DNA typing for applications in forensic science.

Animals↗
PubMed1994

Fluorescence in situ hybridisation of multiple probes on a single microscope slide.

We report a method to analyse multiple samples by fluorescence in situ hybridisation on a single glass microscope slide. Wells were formed in which independent hybridisation reactions could proceed by sealing a silicon rubber gasket to the slide. In the largest format tested, different probes were hybridised simultaneously by applying them directly from a 96-well microtitre dish which was inverted on a glass plate. This technique will increase the rate of analysis of multiple probes against a standard set of chromosomes and could also be used to analyse different karyotypes using a panel of probes such as single chromosome paints during a single operation. It should be useful for both chromosomal mapping projects and screening for chromosome abnormalities in clinical diagnostic laboratories.

Chromosomes↗
PubMed1994

Parallel synthesis and analysis of large numbers of related chemical compounds: applications to oligonucleotides.

This review presents methods for addressing the issue of molecular complexity in biological systems. Biomolecules immobilised on solid phases can be used to probe biological interactions. As a specific example, arrays of large numbers of oligonucleotides allow the analysis of DNA sequences and complex populations of DNA molecules. The specific embodiments of this new method are presented, research up to mid-1992 outlined and requirements for future development discussed.

Base Sequence↗
PubMed1994

Studies of oligonucleotide interactions by hybridisation to arrays: the influence of dangling ends on duplex yield.

Effects of dangling ends on duplex yield have been assessed by hybridisation of oligonucleotides to an array of oligonucleotides synthesised on the surface of a solid support. The array consists of decanucleotides and shorter sequences. One of the decanucleotides in the array was fully complementary to the decanucleotide used as solution target. Others were complementary over seven to nine bases, with overhangs of one to three bases. Duplexes involving different decanucleotides had different overhangs at the 3' and 5' ends. Some duplexes involving shorter oligonucleotides had the same regions of complementarity as these decanucleotides, but with fewer overhanging bases. This analysis allows simultaneous assessment of the effects of differing bases at both 5' and 3' ends of the oligonucleotide in duplexes formed under identical reaction conditions. The results indicate that a 5' overhang is more stabilising than a 3' overhang, which is consistent with previous results obtained with DNA overhangs. However, it is not clear whether this is due to the orientation of the overhang or to the effect of specific bases.

Base Sequence↗
PubMed1994

Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids.

Arrays of oligonucleotides corresponding to a full set of complements of a known sequence can be made in a single series of base couplings in which each base in the complement is added in turn. Coupling is carried out on the surface of a solid support such as a glass plate, using a device which applies reagents in a defined area. The device is displaced by a fixed movement after each coupling reaction so that consecutive couplings overlap only a portion of previous ones. The shape and size of the device and the amount by which it is displaced at each step determines the length of the oligonucleotides. Certain shapes create arrays of oligonucleotides from mononucleotides up to a given length in a single series of couplings. The array is used in a hybridisation reaction to a labelled target sequence, and shows the hybridisation behaviour of every oligonucleotide in the target sequence with its complement in the array. Applications include sequence comparison to test for mutation, analysis of secondary structure, and optimisation of PCR primer and antisense oligonucleotide design.

Base Sequence↗
PubMed1994

Studies on the base pairing properties of deoxyinosine by solid phase hybridisation to oligonucleotides.

Extensive analyses of the base-pairing properties of deoxyinosine to A, C, G, and T were carried out by measuring the hybridisation of oligonucleotides with deoxyinosine in various positions to complementary sets of oligonucleotides made as an array on the surface of a glass microscope slide. With deoxyinosine in internal positions, results are consistent with previous studies, showing a preferential order for pairing of I-C > I-A > I-G approximately I-T. With two adjacent deoxyinosines in the centre of the oligonucleotide, the order in duplex yield is CC > CA > AA > AC > GC > GA > CG > TA > TC > CT = AG > AT > GT > TT. With deoxyinosine at the ends of the oligonucleotide, we find that at the 3' end there is the same order in duplex yield as for the deoxyinosine in internal positions, though with lower discrimination between the bases. When hybridisation is carried out in TMACI there is little base pairing discrimination with deoxyinosine, or indeed any of the four natural bases at the 5' end. Changing the cation to Na+ increased discrimination slightly.

Base Composition↗
PubMed1993

A study of oligonucleotide reassociation using large arrays of oligonucleotides synthesised on a glass support.

An extensive analysis of oligonucleotide interactions was carried out by hybridising a synthetic pool of 256 10mers, A(C,T)8A, representing all oligopyrimidine octamer sequences to an array of four copies of all 256 different octapurine sequences. The resulting 256 duplexes were quantified by phosphorimaging and analysed to determine the dependence of duplex formation on base composition, sequence, and salt concentration. The results show that the base composition dependence of duplex formation can be reduced by high concentrations of tetramethylammonium chloride. This chaotropic solvent also increases duplex yield by up to fifty-fold.

Base Composition↗
PubMed1992

Parallel analysis of oligodeoxyribonucleotide (oligonucleotide) interactions. I. Analysis of factors influencing oligonucleotide duplex formation.

A novel method for the analysis of oligonucleotide-oligonucleotide interactions is described. Oligonucleotides of different sequence are synthesised in situ as stripes on the surface of a glass slide (see accompanying paper). Multiple hybridizations are then carried out on each oligonucleotide simultaneously to determine the dependence of oligonucleotide duplex formation on duplex length, base composition, hybridisation solvent and sequence complexity.

Base Composition↗
PubMed1992

Oligonucleotide hybridizations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesised in situ.

A novel linker for the synthesis of oligonucleotides on a glass support is described. Oligonucleotides synthesised on the support remain tethered to the support after ammonia treatment and are shown to take part in sequence specific hybridisation reactions. These hybridizations were carried out with oligonucleotides synthesised on 'ballotini' solid sphere glass beads and microscope slides. The linker has a hexaethylene glycol spacer, bound to the glass via a glycidoxypropyl silane, terminating in a primary hydroxyl group that serves as starting point for automated or manual oligonucleotide synthesis.

Base Sequence↗
PubMed1992

Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models.

An efficient method was developed for making complete sets of oligonucleotides of defined length, covalently attached to the surface of a glass plate, by synthesizing them in situ. A device carrying all octapurine sequences was used to explore factors affecting molecular hybridization of the tethered oligonucleotides, to develop computer-aided methods for analyzing the data, and to test the feasibility of using the method for sequence analysis. Further development is needed before the method can be used routinely, but our work shows that it has a number of potential advantages over gel-based methods: it should be easy to automate; the quality of the sequence results can be evaluated statistically; it provides a powerful way of comparing related sequences and detecting mutation; it can be applied to both DNA and RNA; and specific motifs can be incorporated into all sequences of the array to focus analysis on sequences of biological interest.

Base Sequence↗