Keith R. Mitchelson

The use of capillary electrophoresis for DNA polymorphism analysis

Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.

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.

Inferring an original sequence from erroneous copies: two approaches

This paper considers the problem of inferring an original sequence from a number of erroneous copies. The problem arises in DNA sequencing, particularly in the context of emerging technologies that provide high throughput or other advantages at the cost of an increased number of errors. We describe and compare two approaches that have recently been developed by the authors. The first approach searches for a sequence known as a Steiner string; the second searches for the most probable original sequence with respect to a simple Bayesian model of sequencing errors. We present the results of extensive tests in which erroneous copies of real DNA sequences were simulated and the algorithms were used to infer the original sequences. The results are used to compare the two approaches to each other and to a third, more conventional, approach based on multiple sequence alignment. We find that the Bayesian approach is superior to the Steiner approach, which in turn is superior to the alignment approach. The two new algorithms can also be used to construct multiple sequence alignments. We show that the two methods produce alignments of approximately equal quality, and conclude that the Steiner approach is better for this purpose because it is faster. Both methods produce better alignments than a well-known multiple sequence alignment package, for the cases tested.

Chapter 10 Sequencing Aided by Mutagenesis Facilitates the De Novo Sequencing of Megabase DNA Fragments by Short Read Lengths

Since the independent invention of DNA sequencing by Sanger and by Gilbert 30 years ago, it has grown from a small scale technique capable of reading several kilobase-pair of sequence per day into todays multibillion dollar industry. This growth has spurred the development of new sequencing technologies that do not involve either electrophoresis or Sanger sequencing chemistries. Sequencing by Synthesis (SBS) involves multiple parallel micro-sequencing addition events occurring on a surface, where data from each round is detected by imaging. New High Throughput Technologies for DNA Sequencing and Genomics is the second volume in the Perspectives in Bioanalysis series, which looks at the electroanalytical chemistry of nucleic acids and proteins, development of electrochemical sensors and their application in biomedicine and in the new fields of genomics and proteomics. The authors have expertly formatted the information for a wide variety of readers, including new developments that will inspire students and young scientists to create new tools for science and medicine in the 21st century. Reviews of complementary developments in Sanger and SBS sequencing chemistries, capillary electrophoresis and microdevice integration, MS sequencing and applications set the framework for the book.

Sequencing by Aligning Mutated DNA Fragments (SAM)

This paper discusses an original technique, Sequencing by Aligning Mutants (SAM), the purpose of which is to overcome sequencing difficulties caused by problematic genomic regions where local sequence characteristics hinder existing sequencing technologies. It involves forming a number of mutated copies of regions of the target DNA, cloning and sequencing each of these mutated fragments. The effectiveness of SAM technology is demonstrated by sequencing of problematic DNA elements. An application of SAM technology to chip-based sequencing-by-hybridization (SBH) is discussed.