Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure.

The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.

Novel site-specific DNA modification in Streptomyces: analysis of preferred intragenic modification sites present in a 5.7 kb amplified DNA sequence.

Both Streptomyces lividans and Streptomyces avermitilis encode similar systems of post-replicative DNA modification which act site-specifically on closely opposed guanines on either strand. The modifications can be detected since they react in vitro with an oxidative derivative of Tris, resulting in strand cleavage. Previous analysis of the preferred modification site of plasmid pIJ101 indicated that extensive amounts of flanking sequence, including direct and inverted repeat structures, are required to direct modification in vivo within a central 6 bp palindrome. We have now examined the preferred modification sites of a chromosomal element, the 5.7 kb amplified DNA sequence (ADS5.7) found in certain S. lividans mutants. In contrast to the pIJ101 site, each of the ADS5. 7sites is intragenic and modified with a 10-fold reduced frequency. However, similar extents of flanking sequence are required for authentic double-strand modification; deletion mutants exhibited different modification profiles, including displaced double-stranded or single-stranded modi-fication. Comparison of different modification sites reveals conservation of the central core sequence, but no significant similarities between flanking sequences. Enhanced modification was detected in a cloned region of the ADS5.7, suggesting that local DNA topology, probably influenced by both DNA supercoiling and the nature of flanking sequences, can influence the modifying activity.

Traumatic ulnar physeal arrest after distal forearm fractures in children.

A review of the orthopaedic literature suggests that traumatic ulnar physeal arrest associated with radial fracture is a rare occurrence. Twenty-three cases of traumatic ulnar physeal arrest have been reported in patients with distal radius fractures, and we report five additional cases. A classification system for the distal ulnar growth deformities is proposed, and the compensatory radial changes are reviewed. Surgical indications for treatment include cosmetic deformity, progressive carpal subluxation, and decreased range of motion. Surgical options for treatment are discussed and include epiphysiodesis, ulnar lengthening, radial osteotomy, and the Suave-Kapandji procedure.

Percutaneous intramedullary fixation of lateral malleolus fractures: technique and report of early results.

Twenty-four patients with Weber B and low Weber C displaced lateral malleolus fractures who underwent closed reduction and percutaneous internal fixation with an intramedullary, fully threaded, self-tapping screw were retrospectively reviewed. Nineteen of these patients were followed for an average of 63.4 weeks. A good radiographic reduction was obtained in 87.5% of patients, a fair reduction in 8.3%, and a poor reduction in 4.2%. The reduction that was obtained was maintained in all patients. Fracture union was achieved in 95.5% of patients, with an average time to union of 8.2 weeks. In all patients the average time to full weight bearing was 6.8 weeks, whereas that in patients with isolated lateral malleolus fractures was 4.5 weeks. There were no deep wound infections or complaints of painful hardware. At latest follow-up, functional results were excellent in 42.1%, good in 42.1%, fair in 5.3%, and poor in 10.5% of patients. If reduction of the lateral malleolus fracture can be obtained in a closed fashion (with the aid of an image intensifier), we believe that fixation may be performed with an axial screw percutaneously. This technique requires minimal soft-tissue dissection, thereby decreasing wound complications and painful hardware sites that are occasionally observed after open techniques. This closed technique also eliminates screw penetration of the ankle joint and damage to the peroneal tendons, which can be risks when a plate or lag screws are employed as internal fixation. Surgical time is also reduced and tourniquet use is optional. If an acceptable reduction cannot be obtained using this technique, open reduction and internal fixation should be performed.