Q & A. [interview

George Oster is Professor of Biophysics, University of California, Berkeley. He received his B.S. at the U.S. Merchant Marine Academy and his Ph.D. at Columbia University. He began his career in biophysics as a postdoc at the Weizmann Institute under Aharon Katchalsky, where his research involved membrane biophysics and irreversible thermodynamics. His concern for environmental issues led him into population biology, which shaded into evolutionary biology and thence to developmental biology, cell biology and, most recently, protein motors and bacterial motility and pattern formation. His tools are mathematics, physics and computer simulation. He is currently a faculty member in the Departments of Molecular and Cellular Biology and the College of Natural Resources at Berkeley.     

High-pressure gel loader for capillary array electrophoresis microchannel plates.

Microfabricated capillary array electrophoresis (microCAE) microchannel plates are the next generation of bioanalytical separation devices. To fully exploit the capabilities of microCAE devices, supporting technology such as robotic sample loading, gel loading, microplate washing, and data analysis must be developed. Here, we describe a device for loading gel into radial capillary array electrophoresis microplates and for plate washing and drying. The microplates are locked into a loading module, and high-pressure helium is used to drive aqueous separation media or wash solutions into the microchannels through fixtures connected to the central anode reservoir. Microplates are rapidly (30 s to 5 min) loaded with separation media, such as 3%-4.8% linear polyacrylamide or 0.7%-3.0% hydroxyethyl cellulose, for electrophoresis. The effective and rapid gel-filling and plate-cleaning methods together with short electrophoretic analysis times (2-30 min) make microCAE systems versatile and powerful nucleic acid analysis platforms.     

Detection of bacterial pathogen DNA using an integrated complementary metal oxide semiconductor microchip system with capillary array electrophoresis

In this paper, we show an integrated complementary metal oxide semiconductor (CMOS)-based microchip system with capillary array electrophoresis (CAE) for the detection of bacterial pathogen amplified by polymerase chain reaction (PCR). In order to demonstrate the efficacy of PCR reaction for the heat-labile toxin producing enterotoxigenic Escherichia coli (E. coli), which causes cholera-like diarrhea, 100 bp DNA ladders were injected along with the PCR product. Poly(vinylpyrrolidone) (PVP) was used as the separation medium and provided separation resolution which was adequate for the identification of PCR product. The miniaturized integrated CMOS microchip system with CAE has excellent advantages over conventional instrumental systems for analysis of bacterial pathogens such as compactness, low cost, high speed, and multiplex capability. Furthermore, the miniaturized integrated CMOS microchip system should be compatible with a variety of microfabricated devices that aim at more rapid and high-throughput analysis.     

DNA sequencing by capillary array electrophoresis with an electric field strength gradient

We examined the effect of an electric field strength gradient on DNA sequencing efficiency using capillary array electrophoresis. Several types of gradients were applied to DNA sequencing and tested in terms of read length and accuracy. Our original method improved the accuracy of DNA sequencing for longer fragments at high temperature.     

DNA sequencing using 96-capillary array electrophoresis

Practical DNA sequencing in a rugged capillary array electrophoresis system coupled directly to 96-well microtiter plates is demonstrated. A CCD detector was used to monitor all capillaries simultaneously with laser-induced fluorescence at 1.75 frames per second. The reconstructed electropherograms show good signal-to-noise ratios and resolution for the entire capillary array. The system used standard dye labeling and image splitting to obtain fluorescence intensities in two wavelength regions to allow calling up to 410 bases for the DNA sequence. The use of a replaceable poly(ethylene oxide) matrix and a protective poly(vinylpyrrolidone) coating allows high separation speed and short turnaround time for high throughput DNA sequencing. Critical evaluation of the system performance over repeated runs with base calling is presented.     

Horace A. Barker (1907-) pioneer of anaerobic metabolism

Dr Horace A. Barker was born and raised in California. He obtained his Ph.D. in chemistry from the University of California, Berkeley in 1933, and became a faculty member at the same campus in 1936. He devoted his research to the study of bacterial metabolism. His contributions include the detailed studies of various aspects of metabolism such as synthesis and oxidation of fatty acids, fermentation of amino acids and purines, and carbohydrate transformations. He isolated and determined the structure and function of some enzymes and coenzymes from bacteria. He also specifically described many anaerobic metabolic pathways. Dr Barker retired in 1976.     

高速DNA序列分析

高速DNA序列分析是人类基因组研究的关键技术．文章对高速DNA序列分析方法如阵列毛细管电泳、超薄层凝胶板电泳、质谱、杂交法、原子探针法、流动单分子荧光检测法等新进展进行了评论．     

Q & A. [interview

Elaine and Gary Ostrander spent their youth in New Jersey and New York before heading to Nebraska for their teen years and eventually Washington State for High School and college, as their father moved around in library administration. Elaine was an undergraduate at the University of Washington, a graduate student at the Oregon Health Sciences University and a postdoc with James Wang at Harvard, studying DNA supercoiling. She next went to Berkeley, where she began the canine genome project, initiating the meiotic linkage map and working on human chromosome 21 at the Lawrence Berkeley National Labs. In 1993 she moved to the Fred Hutchinson Cancer Research Center where she is now a Member of the Divisions of Clinical Research and Human Biology. She is also an Affiliate Professor of Genome Sciences and Biology at the University of Washington, and heads the Program in Genetics at the Hutchinson Center. Gary completed his undergraduate degree in Biology at Seattle University, a M.S. degree at Illinois State University and a Ph.D at the University of Washington in Ocean and Fisheries Science. He went on to be a postdoc in the Department of Pathology at the University of Washington Medical School while being mentored by Senitroh Hakomori of the Fred Hutchinson Cancer Research Center and Eric Holmes of the Pacific Northwest Research Foundation. His work focused on using novel aspects of the biology of fishes to address fundamental questions about cancer. He subsequently held both faculty and administrative positions at Oklahoma State University. Since 1996, he has been at the Johns Hopkins University, where he currently holds academic appointments in the Departments of Biology and Comparative Medicine and is the Associate Provost for Research.     

The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.

Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.     

Sequencing of Gel-Isolated Proteins Using Microblotter Capillary Liquid Chromatography-Electrospray Mass Spectrometry

Enzymatic digests of proteins isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were separated by capillary high-performance liquid chromatography (HPLC). The column eluate was split to an electrospray mass spectrometer on one side and to both a UV detector and a microblotter on the other side. Using the microblotter, the peptides eluted from the column were collected directly onto a polyvinylidene difluoride (PVDF) membrane for Edman sequencing. Thus, a peptide mass map from the mass spectrometric analysis and a prepared PVDF membrane for subsequent Edman sequencing were generated in a single experiment. The addition of molecular mass information to the blotted LC eluate is useful for determining the most important peaks to undergo Edman sequencing. Coupling the capillary HPLC with a microblotter to electrospray mass spectrometry provides an integrated system for separation, collection, and structural analysis of protein digests. It provides high levels of sensitivity, recovery, and convenience for protein characterization. Proteins loaded onto SDS–PAGE at low picomole levels can be analyzed by the new integrated system.     

发展中的DNA测序技术

发展中的ＤＮＡ测序技术赵晓娟刘金毅综述蔡有余琦祖和＊审校（中国医学科学院中国协和医科大学实验动物研究所北京）ＤＮＡ序列分析是基因工程和分子生物学领域最重要的技术之一,是了解基因结构和功能的基础。“人类基因组计划”（ｈｕｍａｎｇｅｎｏｍｅｐｒｏｊｅｃｔ）的实施,有力地推动了高速ＤＮＡ测序技术的发展。除经典的测序方法在技术环节上的不断改进外,近年来发展了一些全新的ＤＮＡ测序方法,如毛细管凝胶电泳...     

DNA sequencing by capillary electrophoresis (review)

DNA sequencing by capillary electrophoresis has been reviewed with an emphasis on progress during the last four years. The effects of sample purification, composition of sieving matrices, electric field strength, temperature, wall coating and DNA labeling on the DNA sequencing performance are discussed. Multicapillary array instrumentation is compared with one-capillary systems. Integrated systems that perform the whole DNA sequencing operation online starting from the DNA amplification through base calling and data processing are discussed.     

A tribute to Thomas Roosevelt Punnett, Jr. (1926-2008)

We honor here Thomas (Tom) Roosevelt Punnett, Jr. (May 25, 1926–July 4, 2008), who was a pioneer of Biology, particularly of biochemistry of plants and algae, having specialized in photosynthesis under Robert Emerson of the University of Illinois at Urbana-Champaign. He did exciting work on regulation and control of various metabolic reactions. He was an innovator and raconteur par excellence, and he prized critical thinking. His enthusiasm for basic science questions was matched by his grasp of their “real-world” implications. His last project was a patent for anaerobic sewage treatment that he hoped would lead to solution of waste disposal and energy creation world wide, including the clean-up of Lake Erie, where he had sailed as a boy. On the personal side, he had a strong sense of morality and a great wit and humor.     

The next generation of exoskeletons: lighter, cheaper devices are in the works

Many researchers and engineers are busy in their laboratories working on devices that will bring mobility to people who have lost function in the lower body due to an accident, stroke, multiple sclerosis, or other disorders. "Several pretty sophisticated exoskeletons are already on the market now, and they are all similar to each other in terms of technologies, but we're not ready to replace the wheelchair yet," said exoskeleton developer Homayoon Kaz Kazerooni, Ph.D., professor of mechanical engineering at the University of California (UC) at Berkeley. "Eventually, we will have devices that are used by individuals on a daily basis to replace wheelchairs but not with the existing technology. We're at the beginning of a much bigger era in exoskeletons."     

Impact of Admission and Cache Replacement Policies on Response Times of Jobs on Data Grids

Caching techniques have been used widely to improve the performance gaps of storage hierarchies in computing systems. Little is known about the impact of policies on the response times of jobs that access and process very large files in data grids, particularly when data and computations on the data have to be co-located on the same host. In data intensive applications that access large data files over wide area network environment, such as data-grids, the combination of policies for job servicing (or scheduling), caching and cache replacement can significantly impact the performance of grid jobs. We present preliminary results of a simulation study that combines an admission policy with a cache replacement policy when servicing jobs submitted to a storage resource manager.The results show that, in comparison to a first come first serve policy, the response times of jobs are significantly improved, for practical limits of disk cache sizes, when the jobs that are back-logged to access the same files are taken into consideration in scheduling the next file to be retrieved into the disk cache. Not only are the response times of jobs improved, but also the metric measures for caching policies, such as the hit ratio and the average cost per retrieval, are improved irrespective of the cache replacement policy used. Ekow Otoo is research staff scientist with the scientific data management group at Lawrence Berkeley National Laboratory, University of California, Berkeley. He received his B.Sc. degree in Electrical Engineering from the University of Science and Technology, Kumasi, Ghana and a post graduate diploma in Computer Science from the University of Ghana, Legon. In 1977, he received his M.Sc. degree in Computer Science from the University of Newcastle Upon Tyne in Britain and his Ph.D. degree in Computer Science from McGill University, Montreal, Canada in 1983. He joined the faculty of the School of Computer Science, Carleton University, in 1983 and from 1987 to 1999, he was a tenured faculty member of the School of Computer Science, Carleton University, Ottawa, Canada. He has served as research consultant to Bell Northern Research, Ottawa, Canada, and as a research project consultant to the GIS Division, Geomatics Canada, Natural Resources Canada, from 1990 to 1998. Ekow Otoo is a member of the ACM and IEEE. His research interests include database management systems, data structures and algorithms, parallel I/O for high performance computing, parallel and distributed computing. Doron Rotem is currently a senior staff scientist and a member of the Data Management group at the Lawrence Berkeley National Lab. His research interests include Grid Computing, Workflow, Scientific Data Management and Paralled and Distributed Computing and Algorithms. He has published over 80 papers in international journals and conferences in these areas. Prior to that, Dr Rotem co-founded and served as a CTO of a startup company, called CommerceRoute, that made software products in the area of workflow and data integration and before that, he was an Associate Professor in the Department of Computer Science, University of Waterloo, Canada. Dr. Rotem holds a B.Sc degree in Mathematics and Statistics from the Hebrew University, Jerusalem, Israel and a Ph.D. in Computer Science from the University of the Witwatersrand, Johannesburg, South Africa. Arie Shoshani is a senior staff scientist at Lawrence Berkeley National Laboratory. He joined LBNL in 1976. He heads the Scientific Data Management Group. He received his Ph.D. from Princeton University in 1969. From 1969 to 1976, he was a researcher at System Development Corporation, where he worked on the Network Control Program for the ARPAnet, distributed databases, database conversion, and natural language interfaces to data management systems. His current areas of work include data models, query languages, temporal data, statistical and scientific database management, storage management on tertiary storage, and grid storage middleware. Arie is also the director of a Scientific Data Management (SDM) Integrated Software Infrastructure Center (ISIC), one of seven centers selected by the SciDAC program at DOE in 2001. In this capacity, he is coordinating the work of collaborators from 4 DOE laboratories and 4 universities (see: http://sdmcenter.lbl.gov). Dr. Shoshani has published over 65 technical papers in refereed journals and conferences, chaired several workshops, conferences, and panels in database management; and served on numerous program committees for various database conferences. He also served as an associate editor for the ACM Transactions on Database Systems. He was elected a member of the VLDB Endowment Board, served as the Publication Board Chairperson for the VLDB Journal, and as the Vice-President of the VLDB Endowment. His home page is http://www.lbl.gov/arie.     

Cassette labeling for facile construction of energy transfer fluorescent primers.

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.     

Microchip electrophoresis: a method for high-speed SNP detection

As a trial practical application, we have applied optimized microfabricated electrophoresis devices, combined with enzymatic mutation detection methods, to the determination of single nucleotide polymorphism (SNP) sites in the p53 suppressor gene. Using clinical samples, we have achieved robust assays with quality factors as good as conventional electrophoresis in ~100 s. This is 10 and 50 times faster than capillary and slab gel electrophoresis, respectively. The method was highly accurate with an average error of mutation site measurement of only ±5 bp. No clean-up of the digestion mixtures was needed prior to injection. This greatly simplifies sample handling relative to capillary instruments, which is important for high-throughput screening applications. Following identification, absolute mutation determination of the screened samples was achieved in a second microdevice optimized for four-color DNA sequencing. Total run time was 25 min in this second device and sequencing data were in full agreement with ABI Prism^® 377 sequencing runs which required 3.5 h. The tandem application of microdevices for location then full characterization of SNPs appears to confirm many of the improvements claimed for future application of microdevices in practical scaled screening for mutational analysis.     

Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis

An integrated system with a nano-reactor for cycle-sequencing reaction coupled to on-line purification and capillary gel electrophoresis has been demonstrated. Fifty nanoliters of reagent solution, which includes dye-labeled terminators, polymerase, BSA and template, was aspirated and mixed with the template inside the nano-reactor followed by cycle-sequencing reaction. The reaction products were then purified by a size-exclusion chromatographic column operated at 50°C followed by room temperature on-line injection of the DNA fragments into a capillary for gel electrophoresis. Over 450 bases of DNA can be separated and identified. As little as 25 nl reagent solution can be used for the cycle-sequencing reaction with a slightly shorter read length. Significant savings on reagent cost is achieved because the remaining stock solution can be reused without contamination. The steps of cycle sequencing, on-line purification, injection, DNA separation, capillary regeneration, gel-filling and fluidic manipulation were performed with complete automation. This system can be readily multiplexed for high-throughput DNA sequencing or PCR analysis directly from templates or even biological materials.     

Energy-transfer cassette labeling for capillary array electrophoresis short tandem repeat DNA fragment sizing.

Energy-transfer (ET) dye-labeled primers significantly improve fluorescent DNA detection because they permit excitation at a single common wavelength and they produce well separated and intense acceptor dye emission. Recently, a new ET cassette technology was developed [Berti, L. et al. (2001) Anal. Biochem. 292, 188-197] that can be used to label any PCR, sequencing, or other primer of interest. In this report we examine the utility of this ET cassette technology by labeling seven different short tandem repeat (STR) specific primers with each of the four ET cassettes and analyzing the PCR products generated on a MegaBACE-1000 capillary array electrophoresis system. More than 60 amplicons were generated and successfully analyzed with the ET cassette-labeled primers. Both forward and reverse primers were labeled for multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling procedure is ideally suited for creating four-color fluorescent ET primers for STR and other DNA assays where large numbers of different loci are analyzed including sequencing, genetic identification, gene mapping, loss of heterozygosity testing, and linkage analysis.     

Digital fragment analysis of short tandem repeats by high‐throughput amplicon sequencing

High‐throughput sequencing has been proposed as a method to genotype microsatellites and overcome the four main technical drawbacks of capillary electrophoresis: amplification artifacts, imprecise sizing, length homoplasy, and limited multiplex capability. The objective of this project was to test a high‐throughput amplicon sequencing approach to fragment analysis of short tandem repeats and characterize its advantages and disadvantages against traditional capillary electrophoresis. We amplified and sequenced 12 muskrat microsatellite loci from 180 muskrat specimens and analyzed the sequencing data for precision of allele calling, propensity for amplification or sequencing artifacts, and for evidence of length homoplasy. Of the 294 total alleles, we detected by sequencing, only 164 alleles would have been detected by capillary electrophoresis as the remaining 130 alleles (44%) would have been hidden by length homoplasy. The ability to detect a greater number of unique alleles resulted in the ability to resolve greater population genetic structure. The primary advantages of fragment analysis by sequencing are the ability to precisely size fragments, resolve length homoplasy, multiplex many individuals and many loci into a single high‐throughput run, and compare data across projects and across laboratories (present and future) with minimal technical calibration. A significant disadvantage of fragment analysis by sequencing is that the method is only practical and cost‐effective when performed on batches of several hundred samples with multiple loci. Future work is needed to optimize throughput while minimizing costs and to update existing microsatellite allele calling and analysis programs to accommodate sequence‐aware microsatellite data.