Direct sequencing of RAPD fragments using 3'-extended oligonucleotide primers and dye terminator cycle-sequencing.

Random amplified polymorphic DNA (RAPD) markers are used widely to develop high resolution genetic maps and for genome fingerprinting. Typically, single oligomers of approximately 10 nucleotides are used to PCR amplify characteristic RAPD marker fragments. We describe an efficient method for the direct end-sequencing of gel-purified RAPD fragments using one primer from a set of four 3'-terminal extended (A, T, C or G) oligonucleotides, identical to the RAPD primer but for the single nucleotide extension. Strand-specific DNA sequence could be independently read from each of the RAPD fragments without recourse to strand separation or fragment cloning. Informative RAPD fragments could be readily converted into mapped STS or SCAR loci using this technology. The 3'-extended primers may also be used to amplify independent genomic RAPD markers.

The use of capillary electrophoresis for point-mutation screening.

Advances in capillary electrophoresis technology over the past three years have been rapid. Capillary electrophoresis offers high-throughput, high-resolution, automatic operation and on-line detection with automatic data acquisition, and this has stimulated its application to the analysis of DNA mutations. Many different PCR-based DNA-mutation assays have been developed for unknown and known mutations. This article compares conventional PCR-based mutation-detection assays with the methods developed for use with capillary electrophoresis. Future trends for mutation detection using capillary electrophoresis are also assessed, with a special emphasis on totally integrated, microchip capillary-electrophoresis-based mutation-detection systems.

Construction of a restriction map and gene map of the lettuce chloroplast small single-copy region using Southern cross-hybridization.

The small single-copy region (SSCR) of the chloroplast genome of many higher plants typically contain ndh genes encoding proteins that share homology with subunits of the respiratory-chain reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase complex of mitochondria. A map of the lettuce chloroplast SSCR has been determined by Southern cross-hybridization, taking advantage of the high degree of homology between a tobacco small single-copy fragment and a corresponding lettuce chloroplast fragment. The gene order of the SSCR of lettuce and tobacco chloroplasts is similar. The cross-hybridization method can rapidly create a primary gene map of unknown chloroplast fragments, thus providing detailed information of the localization and arrangement of genes and conserved open reading frame regions.

The application of capillary electrophoresis for DNA polymorphism analysis.

CE fractions may also be collected and then subjected to additional analysis. Nanoliter fractions containing size or shape fractionated DNA fragments can be collected on moving affinity membranes (125) or into sample chambers (126). The exact timing of the collection steps is achieved by determining the velocity of each individual zone measured between two detection points near the end of the capillary. The DNA samples may subsequently be identified by probe hybridization, or by PCR-linked sequencing. Capillary fractions containing metabolites and derivatives of DNA and small DNA adducts can also be sampled, and then characterized directly by highly sensitive MALDI-TOF atomic analysis (112-118) and ESI-MS (118,119). The automation and integration of PCR and CE analysis (PCR-CE) on a microchip (3-12,96) will also contribute greatly to its adoption as the analysis tool of choice. Significantly, these tools will be applied for DNA sequencing (75,108), for genome mapping (65) and genotyping (42-46), for improved certainty in disease detection (3-6,107,120) and for DNA mutation analysis (2-12,27,58). Recent improvements in the design CAE arrays and associated equipment such as the radial CAE microplate and rotary confocal signal detection system (127) overcome some of the detection limitations of linear CAE and microchip devices and allow the parallel genotyping of 96 samples in about 120 s. The integration of microreactive capillary surface assays (128) and "in-capillary" analysis will also lead to further increases in the speed and sensitivity of CE-based analysis. The recent announcement of the completion of the first draft sequence of the 90% of the entire human genome within 6 mo by Celera Genomics by sequencing random DNA fragments using several hundred ABI 3700 machines (129) illustrates the enormous efficiency realized through the automation of DNA sequencing by CAE. Sequencing was performed at an average rate of approximately 6 x 10(9) bases/yr. The CAE machines will now be employed for a concerted resequencing of genome elements to create an extremely high-density polymorphism map of the entire genome (130). This map will be based principally on single nucleotide polymorphisms, and will catapult human medicine into a new era of closely detailed genetic trait mapping to identify the genetic basis of multi-gene diseases.

Analysis of ligase chain reaction products amplified in a silicon-glass chip using capillary electrophoresis.

Ligase chain reaction (LCR) is a useful molecular technique for detecting known point mutations. We report the first example of the use of a disposable silicon-glass micro-chip for LCR and the first application of capillary electrophoresis (CE) to analyze samples amplified by LCR in a chip. Silicon-glass chips were manufactured using conventional photolithography and anodic bonding. The chips provide three distinct advantages for LCR: excellent thermal conductivity, a micro reaction volume ( < 10 microliters), and reproducible, low-cost manufacturing. Investigation and quantitation of amplification efficiency of LCR in a chip or in a tube requires an analytical technique that is faster and more convenient than the conventional slab gel methods. Slab gel electrophoresis uses relatively large amounts of sample and is labor-intensive and time-consuming, and thus is unsuitable for the separation and detection of LCR products. In contrast CE requires sample volume (original LCR products) of less than 1 microliter and is therefore well-suited to analysis of the micro-volume reaction mixture from chips. We combined CE with a sensitive laser induced fluorescence (LIF) detection system for the rapid separation and quantitative detection of LCR products amplified from the lacI gene in a silicon-glass chip. Comparative studies were made with LCR between tubes and silicon-glass chips. CE-LIF analysis is ideally suited to examination of micro-LCR amplification with high throughput. The technologies may find medical uses in disease diagnosis and research.

Polymerase chain reaction heteroduplex polymorphism analysis by entangled solution capillary electrophoresis.

Heteroduplex DNA polymorphism analysis (HPA) makes use of conformational polymorphisms to alter electrophoretic mobility of fragments and can be used to detect non-restrictable loci. We have developed a novel application of entangled solution capillary electrophoresis (ESCE) to separate heteroduplex and homoduplex DNA molecules. The addition of ethidium bromide and glycerol to the free solution sieving buffer resulted in the improved peak resolution and good reproducibility. Reannealed polymerase chain reaction products could be used directly for mutation screening and with fully automated ESCE the entire HPA may be completed in less than 30 min including sample handling. This technology could provide a rapid and highly efficient way for screening rare mutations among large numbers of individuals.

Antagonism of the Two-Needle Pine Stem Rust Fungi Cronartium flaccidum and Peridermium pini by Cladosporium tenuissimum In Vitro and In Planta.

ABSTRACT Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degrees C. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wall and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host cell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta-1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

Glycerol-enhanced separation of DNA fragments in entangled solution capillary electrophoresis.

(Hydroxyalkyl)methylcellulose additives to borate buffers have been used to create physically entangled solutions for fractionation of biological molecules by high-performance capillary electrophoresis, i.e., ESCE. We have observed that addition of glycerol to an entangled solution containing (hydroxypropyl)methylcellulose (HPMC) in Tris-borate buffer markedly improved the separation of double-strand DNA molecules ranging from less than 100 bp to about 1 kb by ESCE. This range of DNA fragment sizes is well suited to many PCR-based studies. We attribute the improved ESCE performance to the borate present in the buffer system. Borate can form dimeric 1:2 borate:didiol complexes with both glycerol and HPMC, acting as a central linkage to allow an entangled solution with different pore size to form chemically.

Metastable single-strand DNA conformational polymorphism analysis results in enhanced polymorphism detection.

Single-strand DNA conformational polymorphism (SSCP) makes use of sequence-dependent folding of single-stranded DNA (ssDNA), which alters the electrophoretic mobility of the fragments, to detect sequence differences between closely related molecules. In this study ssDNAs were purified by depletion of the complementary strand and PCR reactants on magnetic M-280-strepavidin beads. It was found that SSCP profiles created by purified ssDNAs differ from the profiles created by more usual SSCP methods. Under some conditions, SSCP profiles using whole PCR reaction products may result from the interaction between residual PCR primers and ssDNAs. We observed that the ratio of conformers revealed by band position and band intensity may vary between the assay techniques and misinterpretation of sequence variants may result. Another observation of this study was the formation of metastable conformational isomers with bead-purified ssDNAs by eliminating the thermal treatment used in conventional SSCP methods. The metastable SSCP (mSSCP) represents a novel and sensitive system for detection of sequence variation between closely related DNAs. The technique used here for the preparation of the purified ssDNAs is potentially useful for automated PCR-SSCP analysis using capillary electrophoresis or other methods.

Isolation of a residual protein structure from nuclei of the myxomycete Physarum polycephalum.

A nuclear framework structure has been obtained from isolated interphase nuclei of Physarum polycephalum by extraction with 2.5 M NaCl and subsequent digestion with DNase. Whole-mount electron micrographs showed a nuclear lamina containing residual pore structures associated with the fibrous internal matrix. The matrix was continuous with fibrillar remnants of the nucleolus. The structure was shown to consist of 2 major polypeptides of 23,000 and 36,500 Daltons as well as 30 to 40 minor polypeptides of various molecular weight classes. The 2 major polypeptides were also prominent in preparations of the residual nucleolar material, suggesting that matrix proteins are common to both structures. The predominance of low-molecular-weight polypeptides in Physarum nuclear matrix suggests that there may be significant differences in composition of nuclear structural proteins between lower and higher eukaryotes.