Molecular Biology Current Innovations and Future Trends Part 2 Chapter Abstracts
Chapter 1. AUTOMATED REACTION ASSEMBLY FOR PRODUCTION-SCALE DNA SEQUENCING
Lori A. Weinstock and Elaine R. Mardis
The combination of robotics and molecular biology for high throughput DNA sequencing reaction assembly is an obvious one that has been demonstrated in our laboratory in the context of the Caenorhabditis elegans genome sequencing project. A commercially available pipetting robot, the Hamilton MicroLab 2200, with low volume pipetting capability and sufficient workspace to process up to 1536 cycled sequencing reactions has been modified and used for the process of fluorescent-labeled cycle sequence reaction assembly, ethanol precipitation and pooling. Our report describes detailed protocols for this procedure, including the instrument modifications required to make the procedure robust enough for production scale shotgun sequencing projects.
Chapter 2. HIGH SENSITIVITY PROTEIN SEQUENCE ANALYSIS USING IN SITU PROTEASE DIGESTION ON PVDF MEMBRANES, BIPHASIC CARTRIDGE SEQUENCING AND MALDI MASS SPECTROMETRY
David W. Speicher, David F. Reim, and Kaye D. Speicher
Currently, the most common applications for protein sequence analysis include: 1) determination of partial sequences to either confirm putative clones or to design oligonucleotide probes for screening cDNA libraries; 2) determination of the sites and nature of post-translational modifications implicated in biological function, and 3) characterization of recombinant proteins and fragments of natural or recombinant proteins used in structure-function studies. The most challenging applications are those where only very limited amounts of the desired protein can be isolated. Consequently there is a continuing need to develop higher sensitivity methods for isolation and partial sequence analysis of proteins. N-terminal and/or internal sequences can usually be obtained if at least 50 to 100 pmoles of the protein of interest can be isolated. When less than 10 to 20 pmoles of the desired protein is available, obtaining sequence information is technically feasible, but far from routine for most laboratories. Some of the more important recent advances that contribute to the presently available sequencing sensitivity levels include the use of polyvinylidene difluoride (PVDF) membranes to purify proteins by electroblotting from polyacrylamide gels, improved methods for in situ proteolysis either on blot membranes or in the gel matrix, and the recent availability of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) as a complementary technique to conventional sequence analysis methods. If substantial efforts are devoted to optimizing protein isolation, protein cleavage/peptide purification, and sequence analysis methods, N-terminal sequence data on as little as 5 pmoles of an electroblotted protein or internal sequences from as little as 15 to 20 pmoles of protein can be obtained. Since the N-terminals of the majority of all proteins are naturally blocked, the preferred initial method for obtaining partial protein sequence information of unknown proteins is to obtain internal sequences rather than attempting an N terminal sequence run.
Chapter 3. Electroporation: A Powerful Tool for Introducing Foreign DNA into Eukaryotic and Prokaryotic Cells.
Jhy-Jhu Lin.
Electroporation is a useful technique for the introduction of foreign DNA into both eukaryotic and prokaryotic cells. A significant improvement in the transformation efficiency of Escherichia coli has been achieved using electroporation. In this chapter, the theory of electroporation is addressed with an emphasis on the practical application of electroporation for the introduction of foreign DNA into cells. Protocols for the preparation of electrocompetent E. coli and tobacco plant protoplasts, as well as electroporation conditions are provided. Future applications for electroporation such as the investigation of plasmid DNA replication, expression of multiple plasmids in E. coli, and the establishment of transgenic plants using direct electroporation of foreign DNA into plants tissues or calli are discussed.
Chapter 4. Non-Radioactive Labelling and Detection.
Martin Cunningham, Adrian Simmonds and Ian Durrant.
Procedures for the non-radioactive detection of nucleic acids and proteins have now become standard in many laboratories. Both types of molecule can be efficiently detected on blots using chemiluminescence. Horseradish peroxidase (HRP) catalyses such a reaction involving the oxidation of luminol (the enhanced chemiluminescence reaction) which is widely used in protein detection on Western blots. Following incubation of the blot with a primary antibody, HRP is introduced by means of a secondary antibody-HRP conjugate. Subsequent exposure to X-ray film produces signal within minutes. Nucleic acid probes can be directly labelled with HRP. Following hybridization and stringency washes at 42 ûC, hybrids can be detected with good sensitivity within 60 minutes. Greater sensitivity in nucleic acid detection can be obtained using alkaline phosphatase (AP) catalysed degradation of dioxetane phosphate. Probe is labelled with a hapten such as fluorescein, digoxygenin or biotin by the incorporation of hapten-labelled nucleotides. Hybrids are detected by incubation with an anti-hapten-AP conjugate followed by dioxetane and exposure to film (1-4 hours). Less than 100fg target can be detected on a genomic Southern, equivalent to a single copy gene in less than 1µg mammalian DNA.
Chapter 5. Peptide Nucleic Acid (PNA): A New Molecular Tool.
Peter E. Nielson and Henrik ¯rum.
Synthetic oligonucleotides are indispensable tools in molecular biology being employed as hybridization probes, as primers for PCR and sequencing, for mutagenesis and other gene cloning studies and a variety of other applications.
Much effort has been invested in the development of synthetic oligonucleotide analogues with the goal of obtaining anti-sense drugs with properties superior to those of natural oligo(deoxy)ribonucleotides, in particular with respect to biostability, hybridization efficacy and specificity, and cellular uptake (1, 2). None of these analogues have found their way into general use molecular biology, although phosphorothioates are occasionally being used for anti-sense gene knock-out experiments.
The present chapter is concerned with a special oligonucleotide analogue, PNA (peptide nucleic acid), in which the backbone is a pseudopeptide consisting of N-(2-aminoethyl)glycine units (3-5; Figure 1). Due to the major differences between PNA chemistry and oligonucleotide chemistry, PNA has chemical, physical and biological properties that differ significantly from those of oligonucleotides, although the hybridization efficiency and specificity is retained or even improved. As described below these special properties can be exploited in the development of PNA as biomolecular tools.
Chapter 6. Magnetic Beads in Molecular Biology.
Bjorn-Ivar Haukanes.
Substantial effort has been put into developing methods that enable the separation of specific molecules and cells. The magnetic separation technique using paramagnetic particles with specific active groups at the surface has become a valuable tool during recent years. A specific ligand, with high affinity for the target molecule or cell, is coupled to the activated surface groups of the beads. By means of an external magnet, isolation of the ligand-target molecule or cell complex is simple, fast and less laborious than several other traditional separation techniques. A very specific selection for target is ensured by applying the biotin-streptavidin affinity; using streptavidin as the ligand and biotin as the target molecule. Exploitation of this affinity in particular, has opened the door to the use of magnetic beads as a tool in molecular biology. The present article outlines the possibilities and application of magnetic beads in different areas of molecular biology. This relatively new separation technique has already proven valuable in the generation of single-stranded DNA, in combination with the polymerase chain reaction (PCR), in DNA sequencing, in the purification and identification of specific nucleic acids, in the isolation of DNA binding proteins, and in the fractionation of specific cells and subcellular organelles.
Chapter 7. Antisense Technology and Ribosomes.
D. Castanotto, E. Bertrand and J.J. Rossi.
In recent years significant progress has been made in the use of ribozyme and antisense oligonucleotides for manipulation of gene expression. Ways are being found to increase the efficacy of these molecules in cells, however a number of problems need to be solved before drug technologies for practical treatments of viral and genetic diseases can be developed. The successful use of ribozymes and antisense oligonucleotides will especially depend on a better understanding of the rules governing the intracellular localization, and on the improvement of systems for delivering them to specific cells. This review summarizes the general theory and methodology that stand behind the use and applications of ribozyme and antisense oligonucleotides.
Chapter 8. Methods for Building Phylogenetic Trees of Genes and Species.
Naruya Saitou.
This chapter deals with phylogenetic trees of genes and species, that are fundamental not only for evolutionary studies but for molecular biology in general. The mathematical properties of phylogenetic trees such as the difference between rooted and unrooted trees and the number of possible tree topologies are first explained. Then the biological properties of phylogenetic trees in general is discussed with special reference to the difference between gene trees and species trees. The next section gives description of various tree-building methods such as the unweighted pair group method with arithmetic mean (UPGMA), the neighbor-joining, the maximum parsimony, and the maximum likelihood methods with worked-out examples. Results from computer simulation studies and statistical tests of estimated phylogenetic trees follow. Introduction of various computer packages for tree-building analyses and future trends are given at the end.
Chapter 9. Advances in Nucleic Acid Analysis by HPLC.
Shigeori Takenaka and Hiroki Kondo.
A variety of interactions between nucleic acids and the stationary phase in high-performance liquid chromatography (HPLC) are possible. Many new stationary phases have been developed based on nucleic acid-stationary phase interactions. Herein, we review commonly used techniques including reversed-phase, ion-exchange and size-exclusion chromatography as well as newer techniques including affinity, mixed-mode and slalom chromatography. In addition, we discuss new chromatographic stationary phases which take advantage of hydrogen bonding and aromatic interactions of the bases. These include techniques based on the stacking interaction of bases with the stationary phase or intercalation of groups appended to the stationary phase with the base pairs of double helix DNA. Development of a non-porous anion-exchanger column allows HPLC to replace the gel electrophoretic methods commonly used in molecular biology. Examples of this technology include the quantitation of polymerase chain reaction (PCR) products by HPLC. DNA probe methods by using HPLC with electrochemical detector (ECD) are also discussed.
Chapter 10. Structure Elucidation of Proteins by NMR.
Stefan M. V. Freund and Mark Bycroft.
Structural biology has largely benefited from recent advances in multidimensional NMR spectroscopy: The problem of intrinsic low sensitivity of heteronuclear (15N, 13C) experiments at natural abundance has been solved by genetic engineering techniques, which enable many proteins to be overexpressed and labelled with NMR observable stable isotopes. Multidimensional NMR experiments reduce signal overlap, particularly important for larger proteins, and add non proton dimensions which can circumvent proton signal degeneracy.
Several strategies have been worked out to unambiguously assign proton and /or heteronuclei signals. Based on these assignments structural parameters, such as interproton distances and torsion angles, can be extracted, which are used in NMR restrained molecular modelling to calculate solution structures of proteins. In addition NMR spectroscopy can provide a detailed insight into dynamic phenomena and interactions between biological macromolecules.
Current Books: