Transcripts representing mRNAs of three Xenopus cyclins, B1, B4 and B5, were hybridised to arrays of oligonucleotides scanning the first 120 nt of the coding region to assess the ability of the immobilised oligonucleotides to form heteroduplexes with their targets. Oligonucleotides that produced high heteroduplex yield and others that showed little annealing were assayed for their effect on translation of endogenous cyclin mRNAs in Xenopus egg extracts and their ability to promote cleavage of cyclin mRNAs in oocytes by RNase H. Excellent correlation was found between antisense potency and affinity of oligonucleotides for the cyclin transcripts as measured by the array, despite the complexity of the cellular environment.

Methods are described for the separation of oligodeoxyribonucleotides and oligoribonucleotides by chromatography on thin layers of cellulose impregnated with polyethyleneimine.

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.

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.

Complete libraries of oligonucleotides were used as substrates for Thermus thermophilus DNA ligase, on a M13mp18 ssDNA template. A 17mer primer was used to start a polymerisation process. Ladders of ligation products were analysed by gel electrophoresis. Octa-, nona- and decanucleotide libraries were compared. Nonanucleotides were optimum for polymerisation and up to 15 monomers were ligated. The fidelity of incorporation was studied by sequencing 28 clones (2268 bases) of nonanucleotide polymers, 12 monomers in length. Of the ligated monomers, 79% were the correct complementary sequence. In a total of 57 (2.5%) mispaired bases, there was a strong bias to G.T, G.A, G.G and A.G mismatches. Of the mismatches, 86% were found to be purines on the incoming oligonucleotide, of which 71% were G. There is evidence for clustering of mismatches within specific 9mers and at specific positions within these 9mers. The most frequent mismatches were at the 5'-terminus of the oligonucleotide, followed by the central position. We suggest that sequence selection was imposed by the ligase and not just by base pairing interactions. The ligase directs polymerisation in the 3' to 5' direction which we propose is linked to its role in lagging strand DNA replication.

Oligonucleotide dendrimers were synthesized using a novel phosphoramidite synthon, tris-2,2,2-[3-(4,4'-dimethoxytrityloxy) propyloxymethyl]ethyl- N , N -diisopropylaminocyanethoxy phosphoramidite. Label, incorporated using [gamma-32P]ATP and polynucleotide kinase, was increased in proportion to the number of 5'-ends. There was a similar increase in signal when these multiply labelled oligonucleotides were used as probes to oligonucleotide arrays. A dendrimeric oligonucleotide was used successfully as a primer in the PCR. The strand bearing the dendrimer was resistant to degradation by T7 Gene 6 exonuclease making it easy to convert the double-stranded product of the PCR to a multiply-labelled, single-stranded probe.

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.

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.

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.

Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson-Crick pairing. The secondary conformation of the target may be destabilised to assist its interaction with oligonucleotide probes. To achieve this, we modified a DNA target, which has self-complementary sequence able to form a hairpin loop, by replacing dC with N:4-ethyldeoxycytidine (d(4Et)C), which hybridises specifically with natural dG to give a G:(4Et)C base pair with reduced stability compared to the natural G:C base pair. Substitution by d(4Et)C greatly reduced formation of the target secondary structure. The lower level of secondary structure allowed hybridisation with complementary probes made with natural bases. We confirmed that hybridisation could be further enhanced by modifying the probes with intercalating groups which stabilise the duplex.

DNA dendrimers with two, three, six, nine or 27 arms were reassociated as complementary pairs in solution or with an array of complementary oligonucleotides on a solid support. In all cases, duplex stabilities were greater than those of unbranched molecules of equal length. A theoretical treatment for the process of dissociation of dendrimers explains the major properties of the complexes. The favourable features of DNA dendrimers-their enhanced stability and the simple predictability of their association behaviour-makes them promising as building blocks for the 'bottom up' approach to nano-assembly. These features also suggest applications in oligonucleotide array/DNA chip technology when higher hybridisation temperatures are required, for example, to melt secon-dary structure in the target.

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.

Despite the simplicity of the concept, almost every step in an antisense experiment poses difficulties. Finding a site that is accessible to intermolecular hybridization with complementary nucleic acids is a major problem and determines the success or failure of an antisense experiment. A major determinant of accessibility appears to be the intramolecular folding in mRNAs that renders much of the molecule inaccessible. However, owing to our poor understanding of RNA folding and the mechanisms of heteroduplex formation, theoretical methods have limited use in finding accessible sites. Such methods are unable to address two major considerations when designing an antisense reagent, i.e., which is the most accessible sequence in the target and what length of the reagent works best in terms of activity and specificity. Empirical approaches appear more successful. Of notable interest, and reviewed here, are 'global' methods based on DNA arrays and on mapping of transcripts with RNase H.

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.

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.

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.

Combinatorial libraries of oligonucleotides on beads were synthesised by a split-and-mix strategy using 5'-DMTr- or 5'-Fmoc- nucleoside phosphoramidites. Trityl moieties with different masses were used to encode for the bases coupled at each step in the synthesis of oligonucleotides selected by hybridisation from the libraries. Tags orthogonal to the nucleotides added were produced by coupling amines of different MW to an activated carboxyl group(s) on the trityl moiety. Tags can be released from the support by laser irradiation and measured directly by TOF without matrix. Alternatively, they may be released by an acidic treatment and then analysed by (MA)LDI-TOF.

The large amount of DNA sequence information produced in recent years has created a need for high-throughput methods in biology and genetics. These include sequencing, comparing gene sequences and genotyping. DNA arrays promise a highly parallel means for analysis of DNA that is fast and cost-effective, and offers scope for application to complex systems and processes. Recent years have seen continued transfer of technology from the microelectronics industry. Rapid application of the technology to genotyping, antisense oligonucleotide selection and gene expression analysis has illustrated the general power of this approach.