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.
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.
DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the  sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of ␣ (pol III catalytic subunit),  (sliding clamp processivity factor), and the essential DnaX ( /␥), ␦ and ␦ components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including ␦-␦ interaction, ␦␦ -/␥ complex formation, and ␣-interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50°C, both with regard to initiation complex formation and processive DNA synthesis. The minimal Tth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72°C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event.DNA replication in all biological systems is performed by specialized multiprotein replicases (1, 2). Cellular replicases consist of three major subassemblies: a sliding clamp processivity factor, a clamp loader, and a specialized polymerase. Replicases, especially bacterial replicases, are rapid and processive consistent with the requirement for them to synthesize a several megabase genome from a single origin in less than one hour.In the prototypic Escherichia coli replication system, a key determinant of processive DNA synthesis is the interaction between the  processivity factor and pol III 1 (3, 4). The dimeric  subunit is a bracelet-shaped molecule that clamps around DNA permitting it to rapidly slide along duplex DNA without dissociating (5).  binds to the pol III ␣ subunit through protein-protein contacts preventing the polymerase from dissociating from the template, ensuring high processivity. Efficient loading of the  subunit onto DNA requires ATP-dependent opening and closing of the clamp by the DnaX complex. The DnaX complex contains the essential DnaX, ␦ and ␦Ј subunits plus two ancillary proteins, and (6 -9). The dnaX gene encodes two proteins, ␥ and , by programmed ribosomal frameshifting (10 -15). Both and the shorter ␥ product share ATPbinding domain I, domain II, and domain III that is responsible for DnaX oligomerization, -binding, and binding of ␦-␦Ј (16 -19). contains two unique domains. domain IV forms a link with the DnaB helicase and domain V binds pol III (17,20,21). Pol III consists of ␣, the catalyti...
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