Download or read book High throughput Analysis with 96 capillary Array Electrophoresis and Integrated Sample Preparation for DNA Sequencing Based on Laser Induced Fluorescence Detection written by Gang Xue and published by . This book was released on 2001 with total page 266 pages. Available in PDF, EPUB and Kindle. Book excerpt: The purpose of this research was to improve the fluorescence detection for the multiplexed capillary array electrophoresis, extend its use beyond the genomic analysis, and to develop an integrated micro-sample preparation system for high-throughput DNA sequencing. We first demonstrated multiplexed capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations in a 96-capillary array system with laser-induced fluorescence detection. Migration times of four kinds of fluoresceins and six polyaromatic hydrocarbons (PAHs) are normalized to one of the capillaries using two internal standards. The relative standard deviations (RSD) after normalization are 0.6-1.4% for the fluoresceins and 0.1-1.5% for the PAHs. Quantitative calibration of the separations based on peak areas is also performed, again with substantial improvement over the raw data. This opens up the possibility of performing massively parallel separations for high-throughput chemical analysis for process monitoring, combinatorial synthesis, and clinical diagnosis. We further improved the fluorescence detection by step laser scanning. A computer-controlled galvanometer scanner is adapted for scanning a focused laser beam across a 96-capillary array for laser-induced fluorescence detection. The signal at a single photomultiplier tube is temporally sorted to distinguish among the capillaries. The limit of detection for fluorescein is 3 x 10−11M (S/N=3) for 5-mW of total laser power scanned at 4 Hz. The observed cross-talk among capillaries is 0.2%. A simple two-dimensional laser scanning scheme was also proposed with slight modification. 488 nm and 514 nm laser lines from a single Ar laser were alternatively scanned through the array to achieve two-color basecalling for successful high-throughput DNA sequencing. Finally, we addressed the sample preparation for the high-throughput DNA sequencing. An integrated system of nanoreactor for cycle-sequencing reaction coupled with online CZE purification and CGE DNA sequencing is presented. Less than 100 nl of cycle sequencing premixed reagent solution was hydrodynamically injected into a fused-silica capillary (75 [mu]m i.d.) inside a homemade microthermocycler for successful sample preparation, online purification, sequencing separation, and basecalling. The whole system is readily adaptable to automated multiplex operation for DNA sequencing or PCR analysis.
Download or read book High Throughput Analysis With 96 Capillary Array Electrophoresis and Integrated Sample Preparation for DNA Sequencing Based on Laser Induced Fluorescence Detection written by and published by . This book was released on 2001 with total page 141 pages. Available in PDF, EPUB and Kindle. Book excerpt: The purpose of this research was to improve the fluorescence detection for the multiplexed capillary array electrophoresis, extend its use beyond the genomic analysis, and to develop an integrated micro-sample preparation system for high-throughput DNA sequencing. The authors first demonstrated multiplexed capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations in a 96-capillary array system with laser-induced fluorescence detection. Migration times of four kinds of fluoresceins and six polyaromatic hydrocarbons (PAHs) are normalized to one of the capillaries using two internal standards. The relative standard deviations (RSD) after normalization are 0.6-1.4% for the fluoresceins and 0.1-1.5% for the PAHs. Quantitative calibration of the separations based on peak areas is also performed, again with substantial improvement over the raw data. This opens up the possibility of performing massively parallel separations for high-throughput chemical analysis for process monitoring, combinatorial synthesis, and clinical diagnosis. The authors further improved the fluorescence detection by step laser scanning. A computer-controlled galvanometer scanner is adapted for scanning a focused laser beam across a 96-capillary array for laser-induced fluorescence detection. The signal at a single photomultiplier tube is temporally sorted to distinguish among the capillaries. The limit of detection for fluorescein is 3 x 10-11 M (S/N = 3) for 5-mW of total laser power scanned at 4 Hz. The observed cross-talk among capillaries is 0.2%. Advantages include the efficient utilization of light due to the high duty-cycle of step scan, good detection performance due to the reduction of stray light, ruggedness due to the small mass of the galvanometer mirror, low cost due to the simplicity of components, and flexibility due to the independent paths for excitation and emission.
Download or read book High Throughput Sample Preparation and Analysis for DNA Sequencing PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique written by and published by . This book was released on 2000 with total page 105 pages. Available in PDF, EPUB and Kindle. Book excerpt: Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an attractive alternative to current methods for genetic analysis and disease diagnosis.
Download or read book High Throughput DNA Sequencing by Multiple Capillary Array Electrophoresis Using Replaceable Polymer Solutions and Laser Induced Fluorescence Detection written by Emanuel Carrilho and published by . This book was released on 1997 with total page 510 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book A New Sieving Matrix for DNA Sequencing Genotyping and Mutation Detection and High throughput Genotyping with a 96 capillary Array System written by and published by . This book was released on 1999 with total page 136 pages. Available in PDF, EPUB and Kindle. Book excerpt: Capillary electrophoresis has been widely accepted as a fast separation technique in DNA analysis. In this dissertation, a new sieving matrix is described for DNA analysis, especially DNA sequencing, genetic typing and mutation detection. A high-throughput 96 capillary array electrophoresis system was also demonstrated for simultaneous multiple genotyping. The authors first evaluated the influence of different capillary coatings on the performance of DNA sequencing. A bare capillary was compared with a DB-wax, an FC-coated and a polyvinylpyrrolidone dynamically coated capillary with PEO as sieving matrix. It was found that covalently-coated capillaries had no better performance than bare capillaries while PVP coating provided excellent and reproducible results. The authors also developed a new sieving Matrix for DNA separation based on commercially available poly(vinylpyrrolidone) (PVP). This sieving matrix has a very low viscosity and an excellent self-coating effect. Successful separations were achieved in uncoated capillaries. Sequencing of M13mp18 showed good resolution up to 500 bases in treated PVP solution. Temperature gradient capillary electrophoresis and PVP solution was applied to mutation detection. A heteroduplex sample and a homoduplex reference were injected during a pair of continuous runs. A temperature gradient of 10 C with a ramp of 0.7 C/min was swept throughout the capillary. Detection was accomplished by laser induced fluorescence detection. Mutation detection was performed by comparing the pattern changes between the homoduplex and the heteroduplex samples. High throughput, high detection rate and easy operation were achieved in this system. They further demonstrated fast and reliable genotyping based on CTTv STR system by multiple-capillary array electrophoresis. The PCR products from individuals were mixed with pooled allelic ladder as an absolute standard and coinjected with a 96-vial tray. Simultaneous one-color laser-induced fluorescence detection was achieved by using a CCD camera. The allele peaks for the unknown sample were identified by comparing the normalized peak intensities of the mixtures to those of the pooled ladder by using a straightforward algorithm. An extremely high level of confidence in matching the bands was indicated with negligible cross-talk (
Download or read book Dissertation Abstracts International written by and published by . This book was released on 2004 with total page 692 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Automation and Integration of Multiplexed On line Sample Preparation with Capillary Electrophoresis for DNA Sequencing written by Hongdong Tan and published by . This book was released on 1998 with total page 316 pages. Available in PDF, EPUB and Kindle. Book excerpt: Subsequently, the sequencing ladders are directly loaded into a corresponding size-exclusion chromatographic column operated at 60°C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at 70°C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. DNA sequencing data from this system allow base calling up to 460 bases with accuracy of 98%. Further optimization of the operational conditions and software should extend the read length. The system is scalable to a 96-capillary array and will benefit not only high-speed, high-throughput DNA sequencing but also in genetic typing, population screening and drug discovery.
Download or read book Capillary Gel Electrophoresis written by Andras Guttman and published by Newnes. This book was released on 2021-12-04 with total page 391 pages. Available in PDF, EPUB and Kindle. Book excerpt: Capillary Gel Electrophoresis and Related Microseparation Techniques covers all theoretical and practical aspects of capillary gel electrophoresis. It also provides an excellent overview of the key application areas of nucleic acid, protein and complex carbohydrate analysis, affinity-based methodologies, micropreparative aspects and related microseparation methods. It not only gives readers a better understanding of how to utilize this technology, but also provides insights into how to determine which method will provide the best technical solutions to particular problems. This book can also serve as a textbook for undergraduate and graduate courses in analytical chemistry, analytical biochemistry, molecular biology and biotechnology courses. Covers all theoretical and practical aspects of capillary gel electrophoresis Excellent overview of the key applications of nucleic acid, protein and complex carbohydrate analysis, affinity-based methodologies, micropreparative aspects and related microseparation methods Teaches readers how to use the technology and select methods that are ideal for fundamental problems Can serve as a textbook for undergraduate and graduate courses in analytical chemistry, analytical biochemistry, molecular biology and biotechnology courses
Download or read book Capillary Electrophoresis of Nucleic Acids written by Keith R. Mitchelson and published by Springer Science & Business Media. This book was released on 2008-02-05 with total page 481 pages. Available in PDF, EPUB and Kindle. Book excerpt: The development of PCR, which enables extremely small amounts of DNA to be amplified, led to the rapid development of a multiplicity of a- lytical procedures that permit use of this new resource for the analysis of genetic variation and for the detection of disease-causing mutations. The advent of capillary electrophoresis (CE), with its power to separate and a- lyze very small amounts of DNA, has also stimulated researchers to develop analytical procedures for the CE format. The advantages of CE in terms of speed and reproducibility of analyses are manifold. Furthermore, the high s- sitivity of detection, and the ability to increase sample throughput with par- lel analysis, has led to the creation of a full range of analysis of DNA molecules, from modified DNA adducts and single-strand oligonucleotides through PCR-amplified DNA fragments and whole chromosomes. Capillary Elect- phoresis of Nucleic Acids focuses on analytical protocols that can be used for detection and analysis of mutations and modification, from precise DNA loci through entire genomes of organisms. Important practical considerations for CE, such as the choice of separation media, electrophoresis conditions, and the influence of buffer additives and dyes on DNA mobility, are discussed in several key chapters and within particular applications.
Download or read book Analytical Techniques In DNA Sequencing written by Brian K. Nunnally and published by CRC Press. This book was released on 2005-06-23 with total page 252 pages. Available in PDF, EPUB and Kindle. Book excerpt: While one marvels at the DNA generated by the Human Genome Project, perhaps just as significant and fascinating is the development of the amazing analytical technology that has permitted us entry into the genomic era and a whole new level of scientific understanding. Analytical Techniques in DNA Sequencing takes a look at the various DNA
Download or read book Molecular Diagnostics written by George P. Patrinos and published by Academic Press. This book was released on 2016-10-27 with total page 526 pages. Available in PDF, EPUB and Kindle. Book excerpt: Molecular Diagnostics, Third Edition, focuses on the technologies and applications that professionals need to work in, develop, and manage a clinical diagnostic laboratory. Each chapter contains an expert introduction to each subject that is next to technical details and many applications for molecular genetic testing that can be found in comprehensive reference lists at the end of each chapter. Contents are divided into three parts, technologies, application of those technologies, and related issues. The first part is dedicated to the battery of the most widely used molecular pathology techniques. New chapters have been added, including the various new technologies involved in next-generation sequencing (mutation detection, gene expression, etc.), mass spectrometry, and protein-specific methodologies. All revised chapters have been completely updated, to include not only technology innovations, but also novel diagnostic applications. As with previous editions, each of the chapters in this section includes a brief description of the technique followed by examples from the area of expertise from the selected contributor. The second part of the book attempts to integrate previously analyzed technologies into the different aspects of molecular diagnostics, such as identification of genetically modified organisms, stem cells, pharmacogenomics, modern forensic science, molecular microbiology, and genetic diagnosis. Part three focuses on various everyday issues in a diagnostic laboratory, from genetic counseling and related ethical and psychological issues, to safety and quality management. Presents a comprehensive account of all new technologies and applications used in clinical diagnostic laboratories Explores a wide range of molecular-based tests that are available to assess DNA variation and changes in gene expression Offers clear translational presentations by the top molecular pathologists, clinical chemists, and molecular geneticists in the field
Download or read book Medical BioMethods Handbook written by John M. Walker and published by Springer Science & Business Media. This book was released on 2007-10-09 with total page 635 pages. Available in PDF, EPUB and Kindle. Book excerpt: John Walker and Ralph Rapley have collected a wide-ranging group of molecular and biochemical techniques that are the most frequently used in medical and clinical research, especially diagnostics. The authors-well-established investigators who run their own research programs and use the methods on a regular basis-outline the practical procedures for using them and describe a variety of pertinent applications. Among the technologies presented are southern and western blotting, electrophoresis, PCR, cDNA and protein microarrays, liquid chromatography, in situ hybridization, karyotyping, flow cytometry, bioinformatics, genomics, and ribotyping. The applications include assays for mutation detection, mRNA analysis, chromosome translocations, inborn errors of metabolism, protein therapeutics, and gene therapy.
Download or read book American Doctoral Dissertations written by and published by . This book was released on 2001 with total page 776 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book An Integrated Multiple Capillary Array Electrophoresis System for High throughput DNA Sequencing written by and published by . This book was released on 1998 with total page 158 pages. Available in PDF, EPUB and Kindle. Book excerpt: A capillary array electrophoresis system was chosen to perform DNA sequencing because of several advantages such as rapid heat dissipation, multiplexing capabilities, gel matrix filling simplicity, and the mature nature of the associated manufacturing technologies. There are two major concerns for the multiple capillary systems. One concern is inter-capillary cross-talk, and the other concern is excitation and detection efficiency. Cross-talk is eliminated through proper optical coupling, good focusing and immersing capillary array into index matching fluid. A side-entry excitation scheme with orthogonal detection was established for large capillary array. Two 100 capillary array formats were used for DNA sequencing. One format is cylindrical capillary with 150 [mu]m o.d., 75 [mu]m i.d and the other format is square capillary with 300 [mu]m out edge and 75 [mu]m inner edge. This project is focused on the development of excitation and detection of DNA as well as performing DNA sequencing. The DNA injection schemes are discussed for the cases of single and bundled capillaries. An individual sampling device was designed. The base-calling was performed for a capillary from the capillary array with the accuracy of 98%.
Download or read book High Throughput Sample Preparation and Analysis for DNA Sequencing PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique written by Yonghua Zhang and published by . This book was released on 2000 with total page 196 pages. Available in PDF, EPUB and Kindle. Book excerpt: After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixture was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis based simply on multiplexed UV imaging absorption detection to increase the throughput. We further developed a new methodology-nonaqueous capillary array electrophoresis coupled with microreaction, to address the throughput needs of combinatorial approaches to homogeneous catalysis screening and reaction optimization. Samples were injected directly from reaction vial without dilution and reaction quenching. Buffer compatibility was also found important for reliable 96-capillary array injection. By choosing deferent Pd and base, a combination of 88 different reaction conditions was quickly tested. The analysis time was less than one minute for one sample.
Download or read book A New Sieving Matrix for DNA Sequencing Genotyping and Mutation Detection and High throughput Genotyping with a 96 capillary Array System written by Qiufeng Gao and published by . This book was released on 1999 with total page 264 pages. Available in PDF, EPUB and Kindle. Book excerpt: A temperature gradient of 10 0C with a ramp of 0.7 0C/min was swept throughout the capillary. Detection was accomplished by laser induced fluorescence detection. Mutation detection was performed by comparing the pattern changes between the homoduplex and the heteroduplex samples. High throughput, high detection rate and easy operation were achieved in this system. We further demonstrated fast and reliable genotyping based on CTTv STR system by multiple-capillary array electrophoresis. The PCR products from individuals were mixed with pooled allelic ladder as an absolute standard and coinjected from a 96-vial tray. Simultaneous one-color laser-induced fluorescence detection was achieved by using a CCD camera. The allele peaks for the unknown sample were identified by comparing the normalized peak intensities of the mixtures to those of the pooled ladder by using a straightforward algorithm. An extremely high level of confidence in matching the bands was indicated with negligible cross-talk (<0.89%) between adjacent capillaries.
Download or read book Commencement written by Iowa State University and published by . This book was released on 2001 with total page 562 pages. Available in PDF, EPUB and Kindle. Book excerpt:
