Abstract
No ABSTRACT is available. The first paragraph of the chapter is as follows......
Genomic technologies are as old as molecular biology. They are probably best defined as technologies used to manipulate and analyze genomic information. With this definition it is clear that the evolution of this collective power began in earnest with the invention of DNA cloning in the 1970's. While the origins of many technical advances discussed in this book find roots in the pre-cloning era, most of the technology derives from the last quarter of the 20th century. As the era of "new biology" emerges with an abundance of genomic sequence information, the historical impact of these technologies is clearly immense. When we realize, however, how many times this label has been applied over the past 40 years, it is obvious that this is merely a reflection of the rapid pace of revolutionary advances, new technologies, experimental and theoretical methods and additional knowledge generated over this entire time. There was even a Nature journal in the 1960s called "Nature New Biology", which was devoted to that very new "bastard" science known as molecular biology. In fact, biology has been reinvented many times over the past 40 years, and the past 5 years have been no exception.
Chapter 2
Molecular Cytogenetics and Human Genome Function
Pierre P. Massion and Joe W. Gray
Abstract
Molecular cytogenetic analyses contribute to the functional analysis of the human and other complex genomes by providing information about the physical organization of genes and genomic segments in metaphase chromosomes and interphase nuclei and by detecting and localizing genomic abnormalities such as amplifications, structural rearrangements and deletions that alter cellular or organism phenotypes. Fluorescence in situ hybridization (FISH) is particularly useful in analyses of genomic organization and copy number in individual cells while comparative genomic hybridization (CGH) allows genome copy number changes that occur in most or all of the cells of a population to be readily associated with genomic sequence. This chapter reviews ways in which FISH and CGH facilitate genomic analyses.
Chapter 3
Genome Comparison Techniques
Lisa Stubbs
Abstract
The shared evolutionary heritage that links different animals, plants and microbes has left its clear genetic imprint, in the form of genomes that are highly similar in gene content, structure, and function. The preservation of functional elements within evolutionarily related genomes has added an additional force to the power to the completed genome sequences of humans, flies, microbes, and other species, since functional and structural information can be extrapolated to other genomes from similar branches of the evolutionary tree. Resources such as gene-based comparative maps, ESTs, and BAC end sequences from many additional species are providing the essential links to facilitate those extrapolations. The large-scale comparisons completed so far have highlighted the power of sequence alignments in identification of genes, regulatory elements, and other functional sequences, providing molecular access to exciting new avenues of genetic research. Comparative sequence alignments have also permitted the differences between genomes to be highlighted, including the gain and loss of genes through duplications, deletions, gene transfer (in microbes) and sequence drift. This chapter will explore the methods for genome comparison, and summarize the highlights of the most illustrative comparative genomics studies completed to date.
Chapter 4
Impact of Transgenic Technologies on Functional Genomics
Cooduvalli S. Shashikant and Frank H. Ruddle
Abstract
Gene transfer technologies in mammals are the focus of renewed interest owing to the recent emphasis on analyzing gene function in the postgenomic era. Three important developments in this area include transgenics, gene targeting and nuclear transfer or animal cloning. These technological have enhanced our ability to analyze gene function at the level of whole organism and provided means to modify gene expressions in. This chapter reviews origins and current status of transgenic technologies. Various applications technologies including chromosome engineering, stem cells, gene traps and modifying livestock are presented. The impact of mouse technologies and genomics on functional analyses are also discussed.
Chapter 5
Present and Future Tags and Labels in High Throughput Genomic Analysis
Jeffrey Van Ness
Abstract
The methods that scientists use to analyze nucleic acids is changing in scale and complexity on almost a yearly basis. What do researchers in both academic and industrial circumstances want in terms of nucleic acid-based assays? Most would agree on the following parameters: 1) low cost, 2) accuracy, 3) ease of use, 4) homogeneous assay format, 5) scalability, 6) robustness and 7) ability to multiplex. In order to achieve these goals the field encompassing genomic technologies is currently undergoing a transformation. Recent advances in instrumentation are allowing the analog type measurements of gels to be replaced with digital measurements amenable to high- throughput assays and analysis. The resulting improvements in throughput enables an unprecedented increase in the scale of measurements that can be made. For example, the average publication appearing in the American Journal of Human Genetics in which vulnerability genes are mapped require about 1 million genotypes. The array of technologies that can currently be employed for making nucleic acid-based measurements is bewildering. To help analyze these diverse technologies, tagging and labeling schemes are discussed separately from the assays and detection systems used in genetic analysis.
Chapter 6
MAGIChip: Properties and Applications in Genomic Studies
Alexander Kolchinsky and Andrei Mirzabekov
Abstract
MAGIChip (Micro-Arrays of Gel-Immobilized Compounds on a Chip) consists of an array of blocks of polyacrylamide gel attached to hydrophobic glass surface. The gel pads range in size from picoliters to nanoliters and are used for immobilization of oligonucleotide probes as well as miniature test tubes for chemical or enzymatic reactions with tethered compounds. Nucleic acids are hybridized, fractionated, and modified inside the pads. Most importantly, all steps of sequence analysis PCR-amplification, detachment of primers and products from the substrate, hybridization, and reading of the results are performed within the same pad. We developed a versatile, flexible, and inexpensive technology platform to monitor processes in the arrays in both real time and steady-state. This platform offers unique possibilities for research and biomedical applications. Various kinetic and thermodynamic analyses of molecular interactions were performed in these settings. Microsequencing on MAGIChips is carried out using fluorescent labels in combination with microscopy or scanning. Stacking interactions with short oligonucleotides enhance the sequencing capabilities of the chips and allow employing MALDI mass spectroscopy for the analysis of the results. Customized MAGIChips were successfully used for screening of SNPs in a broad range of biologically meaningful genes, screening for toxin and drug resistance genes, identification of bacteria and viruses, and studies of rearrangements in human chromosomes.
Chapter 7
Mass Spectrometry of Nucleic Acids
Zhaojing Meng and Patrick A. Limbach
Abstract
Mass spectrometry is a powerful tool for the characterization of nucleotides, oligonucleotides and nucleic acids. The advantages of mass spectrometry for oligonucleotide analysis include high sensitivity, accurate molecular weight determinations and the ability to derive structural information. Historically oligonucleotides and nucleic acids have proven difficult to characterize using mass spectrometry. Recent advances in the development of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) now permit the routine analysis of oligonucleotides and intact nucleic acids. ESI- and MALDI-MS have been used to provide sequence information for small (n <= 50-mers) oligonucleotides. MALDI-MS has been used for single nucleotide polymorphism (SNP) analysis and both methods have been used for the analysis of short tandem repeats (STRs). Finally, MALDI-MS continues to be a promising alternative for high-throughput DNA sequencing, although the present read-lengths are still nearly an order of magnitude less than those of competing technologies. It is predicted that continued developments in methodology and instrumentation will further improve the capabilities of mass spectrometry for nucleic acid analysis.
Chapter 8
Detection of Single Nucleotide Polymorphisms
Pui-Yan Kwok
Abstract
Single nucleotide polymorphism (SNP) detection technologies are used to scan for new polymorphisms and to determine the allele(s) of a known polymorphism in target sequences. SNP detection technologies have evolved from labor intensive, time consuming, and expensive processes to some of the most highly automated, efficient, and relatively inexpensive methods. Driven by the Human Genome Project, these technologies are now maturing and robust strategies are found in both SNP discovery and genotyping areas. The nearly completed human genome sequence provides the reference against which all other sequencing data can be compared. Global SNP discovery is therefore only limited by the amount of funding available for the activity. Local, target, SNP discovery relies mostly on direct DNA sequencing or on denaturing high performance liquid chromatography (dHPLC). The number of SNP genotyping methods has exploded in recent years and many robust methods are currently available. The demand for SNP genotyping is great, however, and no one method is able to meet the needs of all studies using SNPs. Despite the considerable gains over the last decade, new approaches must be developed to lower the cost and increase the speed of SNP detection.
Chapter 9
Live Cell Assays: Tools for Functional Genomics
Gregory W. Henkel
Abstract
Public and private databases are loaded with genetic information thanks to the large scale sequencing efforts of entire genomes over the last decade. The next daunting task is to try and make sense of all this information, to discover the function of not only each gene but how the genome as a whole functions under various conditions. Assays performed in cells have become an important means for studying physiological functions in vivo because they provide the necessary biological mix for many complex processes. In addition, the current state-of-the-art in optical indicators, especially fluorescent molecules, have enabled researchers to devise experimental methods to examine physiological and biochemical activities in living cells. This chapter will review the types of optical indicators used for live cell studies and how they are applied across a variety of cell based assays.
Chapter 10
Automation and Robotics in Genomics Laboratories
Theodore E. Mifflin, Steven D. Hamilton, Gary W. Kramer, and Robin A. Felder
Abstract
Automation and robotics are changing labor-intensive laboratories into streamlined process environments that produce error-free results at a rate that exceeds human capabilities. The task to sequence fully the DNA in a living organism has provided the incentive to create novel high efficiency robotic systems capable of performing laboratory procedures. Automation has streamlined genomics laboratories into facilities where there is routine contamination-free specimen preparation, rapid genome sequencing, and automated data reduction. The first automated laboratories used human-scale robot arms to replicate the movements of humans ("The One-Armed Chedmist"). However, it became quickly apparent that procedures could be modified to take advantage of the super-human capabilities of robotics. Thus, subsequent automated laboratories have used integrated fixed automation capable of performing well-defined procedures with high efficiency. Genome mapping laboratories, as well as clinical molecular diagnostic laboratories, are becoming fully automated using procedures and tools that will be summarized in this chapter. In the future, automation will become more miniaturized to increase speed, reduce costs, and take up less space than conventional automated laboratories.
Chapter 11
Computational DNA Sequence Analysis and Annotation
Hyatt, D. and Uberbacher, E.C.
Abstract
With the explosive growth of genomic and proteomic sequence data, it has become impractical to analyze DNA sequences by purely experimental means. Many biologists routinely use computational tools to predict putative features within their query sequences. This chapter details the computational methods most commonly used by these programs. In addition, this chapter examines the challenges of applying computational methods to the analysis of whole genomes. The particular problem of building a comprehensive system to analyze, store, and visualize annotation data for an entire set of genomes is examined in detail.
Chapter 12
Beyond Sequence Similarity, or Sequence Analysis in the Age of the Genome
Itai Yanai, Adnan Derti and Charles DeLisi
Abstract
Until recently, the only computational method available for predicting the function of an uncharacterized gene was sequence similarity, an approach that is effective but restricted to instances in which the function of a closely related sequence is known. Because homologs of characterized function are not available for many sequenced genes, new methods that do not rely upon sequence similarity are critical if we are to exploit and address the avalanche of sequences. Such methods have in fact emerged recently. By considering the genome as a parts list, we can link two genes functionally if they share the same evolutionary pattern, such that they are either both present or absent in any of the known genomes. Insofar as the genome is a permutation of genes, two genes may be associated if they are consistently found as chromosomal neighbors across genomes. Genes may also be linked if they are found fused as one gene in another genome or if they have common regulatory elements and/or similar expression patterns. Together, these methods constitute the many diverse senses of sequence analysis, which may collectively hint at the function of an uncharacterized sequence in the context of all other known sequences.
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