Molecular Biology Current Innovations and Future Trends. Part 1
Chapter Abstracts
Chapter 1. Recent Advances in PCR Methodology.
Michael P. Weiner, Tim Gackstetter, Gina L. Costa, John C. Bauer, and Keith A. Kretz
Protocols are presented for several PCR methods. These include Pfu DNA polymerase polishing to increase PCR cloning efficiency, blunt-ended PCR cloning, site-directed mutagenesis and colony PCR. The objective of all of these methods when used with PCR is to decrease both the time and the effort involved in routine laboratory procedures. A discussion of laboratory equipment and supplies, including thermocyclers and imaging equipment is included. We also present a brief discussion of the different polymerases used for PCR, including mixed polymerases used for PCR of long regions of DNA.
Chapter 2. Thermal Cycle Sequencing.
Keith A. Kretz
The acquisition of DNA sequence information is an integral part of many research projects. The goals of the Human Genome Project alone include determining the sequence of the 3 billion base pairs of DNA in the human genome as well as several model organisms. To that end a number of improvements have been made in sequencing strategies and data acquisition which allow sequence data to be obtained more efficiently. One of these improvements was the development of thermal cycle sequencing, or linear amplification sequencing, as it is sometimes known. This method utilizes a thermostable DNA polymerase in a temperature cycling format to perform multiple rounds of dideoxynucleotide sequencing on the template. The advantages include stronger signals from smaller amounts of template and the ability to sequence previously unreliable templates.
Chapter 3. Mini-Prep Plasmid DNA Isolation and Purification using Silica-Based Resins.
Paul N. Hengen
It has been common practice in the past to use large quantities of starting material for experimental molecular biology techniques; however, rapid advancements in subcloning and sequencing procedures on a mini-scale have shown that many of these methods no longer require a substantial amount of purified DNA. Due to the high quality of many commercially available purified enzymes and reagents, and the development of many new mini-isolation techniques and prepackaged kits, high quality mini-prep plasmid DNA can now be had for far less investment of time, labour, and materials. A significant reduction in the time researchers spend on purifying their DNA samples has led to an increase in productivity for experimental biologists, one testimony to this being the rapid appearance and availability of numerous new cloning vectors. In this chapter, the different techniques for the isolation and purification of mini-prep plasmid DNA are reviewed and attention is focused on a rapid and inexpensive method utilizing the binding of DNA to diatomaceous earth and other silicates.
Chapter 4. Gel Electrophoresis of DNA and Proteins: Recent Advances in Theory and Practical Applications.
Branko Kozulic
In order to explain the mechanism of electrophoretic migration of macromolecules through gels, it is necessary to define the spaces occupied by the migrating molecules and to describe how the molecules pass through these spaces. Some models of gel electrophoresis consider that the spaces (pores) exist regardless of the presence of the migrating macromolecules, whereas others propose that the migrating molecules influence or create these spaces. The gel polymers are treated as fixed obstacles or displaceable, elastic chains. The present models of gel electrophoresis are compared and their predictions correlated to the published experimental data. Recent progress in the practical applications of gel electrophoresis is briefly reviewed, with the emphasis on emerging techniques and on the useful, but less frequently employed procedures.
Chapter 5. Pulsed Field Gel Electrophoresis.
Alexander Kolchinsky and Roel Funke
Pulsed field gel electrophoresis extends the size range of resolution of DNA molecules to many megabases. This bridges a gap that existed in techniques for the structural analysis of large genomes. The ability to analyze large molecules of DNA has enabled the development of cloning vectors with large capacities such as yeast artificial chromosomes, and permitted applications including the transfer of artificial chromosomes containing intact gene clusters to mammalian cells. It has also brought the creation of artificial chromosomes for mammals and plants into the realm of feasibility.
Chapter 6. Automated DNA Hybridization and Detection.
Stephan Beck
The specific identification of DNA molecules by hybridization with probes complementary to their target sequences has wide ranging applications in research, healthcare and industry. The simplicity of the hybridization procedure in combination with advanced detection systems is well suited for processing large numbers of samples and, with various genome projects gathering pace, DNA hybridization and detection have now become a major focus for automation. Here, the current status and future trends in this field of research will be reviewed and potential applications will be discussed using genome analysis as example.
Chapter 7. An Improved Subtractive Hybridization Method using Phagemid Vectors.
Christian E. Gruber and Wu-Bo Li
Subtractive hybridization is a valuable tool which can be used to identify and enrich genes that are differentially expressed in different cell types, different developmental stages, pathological conditions, or in response to stimuli (i.e. fibroblast factor). Subtractive hybridization is defined as the hybridization between two DNA (or RNA) populations that are closely related, and the removal of the hybridized sequences common to both tissues (or cells). Subsequently, the unhybridized sequences can be preserved as a subtracted cDNA library.
Our protocol uses a biotinylated RNA driver produced from double-stranded pSPORT 2 cDNA plasmid and a single-stranded (ss) pSPORT 1-cDNA target plasmid DNA spiked with small amounts (1:2000 transformation ratio) of pBR322 plasmid DNA. Since this reporter plasmid should contain no complementary sequences to cDNA, it can be used to measure the subtraction efficiency. Alternatively, if the pBR322 plasmid is a concern, a parallel subtraction reaction can be performed without this reporter plasmid. The biotinylated driver RNA is hybridized to target, the common sequences subtracted and pBR322 and specific sequences are enriched. As another option, the biotinylated RNA driver can be produced from pSPORT 1-cDNA and ss DNA target can be prepared from pSPORT 2-cDNA. In this case, linearize the pSPORT 1 with Not I and transcribe with T7 RNA Polymerase to make driver RNA. The ss pSPORT 2-cDNA poly dT region must be blocked with poly dA40-60. In addition to the above mentioned advantages of our system, it is easy to obtain miligram quantities of biotinylated driver RNA by in vitro transcription. Therefore, very concentrated driver can be added to the subtraction reaction, which will more easily drive the hybridization to completion. Finally, we utilize RubensteinŐs ds conversion procedure to improve the cloning efficiency of the unhybridized ss target DNA.
Chapter 8. Oligoribonucleotides: Theory and Synthesis.
Ravi Vinayak
Chemical synthesis of biologically active oligoribonucleotides (RNA) fills a unique role for precise nucleotide substitution and large scale requirements for physical studies, antisense and ribozyme investigations. The growing number of applications for synthetic RNA have generated a demand for more effective methods for synthesis and post-synthesis protocols. In this chapter, chemical synthesis of oligoribonucleotides based on solid-phase chemistry using cyanoethyl phosphoramidites is detailed. Improved post-synthesis protocols are presented that assure isolation of high-quality synthetic RNA. Also, reliable techniques of analysis and purification, based on reverse phase and anion exchange HPLC are described.
Chapter 9. Isoelectric Focusing of Proteins by Capillary Electrophoresis.
Tom Pritchett
Isoelectric focusing has become an important technique for protein chemists and molecular biologists in academic research, biotechnology R&D, and biotechnology quality control laboratories. Isoelectric focusing in capillaries (CIEF) is a relatively new application of capillary electrophoresis which offers several advantages over traditional slab gel techniques. These include direct detection by U.V. absorbance (eliminating laborious staining and de staining), faster single-sample analysis, ease of automation, direct transfer of data to a computerized data station for analysis and storage, and the ability to perform fully quantitative analysis. Resolution is comparable to that achieved using traditional techniques involving carrier ampholytes, but has not yet approached that achieved using immobilized ampholytes.
Chapter 10. DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry.
Ivo G. Gut and Stephan Beck
Current progress towards the application of matrix assisted laser desorption ionization mass spectrometry for DNA analysis is reviewed. The instrumental set-up is described including recent additions that contribute to improved detection. First applications, such as the analysis of short oligonucleotides for quality control are described. The future of the technique and how it might be used as a substitute for fluorescence automated DNA sequencing are outlined.
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