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RECENTLY PUBLISHED:
UNIT 2.10 High-Throughput Genotyping Using the TaqMan Assay (Koustubh Ranada, Bristol-Myers Squibb, Princeton, New Jersey). The failure of linkage analysis to identify, in convincing way, loci for complex diseases has led to a renewed interest in association studies for mapping genes for such traits. For large-scale association studies to become a reality, high-throughput genotyping methods that are flexible, accurate, and employ uniform conditions for typing different SNPs will be required. The TaqMan method described in this unit incorporates allele-specific probes in the PCR combines the amplification and detection steps and requires no post-PCR processing for determining genotypes—for each reaction, fluorescence serves as an indicator and genotypes are inferred based on the values obtained form the measurement.
UNIT 5.1 Pulsed-Field Gel Electrophoresis for Long-Range Restriction Mapping (Robert M. Gemmill, Eleanor Roosevelt Institute for Cancer Research, Boulder, Colorado; Richard Bolin, Nexagen, Boulder, Colorado, Hans Albertsen, Eccles Institute of Human Genetics, Salt Lake City, Utah; and Jeff P. Tomkins and Rod A. Wing, Clemson University Genomics Institute, Clemson, South Carolina.) This unit describes procedures for generating long-range restriction maps of genomic DNA and techniques for analysis of large-insert clones. Instructions for digesting agarose-embedded DNA, PFGE separation, and Southern blotting and hybridization are highlighted. Support protocols for preparation of high-molecular-weight genomic DNA in agarose blocks and agarose microbeads are described, as is the preparation of size standards from l phage, S. cerevisiae, and S. pombe. A new protocol on preparation of BAC DNA, suitable for digestion, mapping, and sequencing, is included in this update.
UNIT 13.1 Gene Delivery to Arteries (Levent M. Akyurek, Hong San, and Elizabeth G. Nabel, National Heart, Blood, and Lung Institute, NIH, Bethesda; and Ripudamanjit Singh and Robert D. Simari, Mayo Clinic and Foundation, Rochester, Minnesota). Introducing recombinant genes into normal, injured, or atherosclerotic arteries is a method used to study modification of native and foreign gene expression in normal and diseased arteries. The original version of this unit included protocols on gene transfer in the ilieofemoral arteries of pigs and rabbits. The update also has these procedures, but additionally includes techniques for the delivery of genes into normal and injured murine carotid and femoral arteries.
UNIT 13.4 Gene Delivery to Muscle (Matthew L. Springer, Thomas A. Rando, and Helen M. Blau, Stanford Universoity School of Medicine, Stanford, California). The delivery of genes to skeletal muscle by myoblast implantation, DNA injection, or viral transduction has therapeutic applications for human neuromuscular and systemic disorders, many of which are now represented by transgenic (or "knock-out") mouse models. This unit describes some of the techniques for the isolation and retroviral transduction of mouse myoblasts, the injection of myoblasts and plasmid DNA into mouse muscle, and the histological assessment of the recipient muscle. The entire unit has been updated to reflect the latest techniques and practices for these procedures; in addition, a protocol on isolating myoblasts form mixed cell populations using cell-sorting techniques has been added.
UNIT 5.12 Introduction of Large Insert DNA into Mammalian Cells and Mouse Embryos (Roger Reeves, Johns Hopkins University School of Medicine). In addition to protocols describing the introduction of YAC clones into mammalian cells, an new protocol on the preparation of BAC and PAC DNA for pronuclear microinjection into mouse embryos has been included in this update.
UNIT 7.12 Single Strand Conformation Polymorphism Analysis Using Capillary Electrophoresis (Lars Allan Larsen, Michael Christiansen, Jens Vuust, and Paal Skytt Andersen, Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark). Single strand conformation polymorphism (SSCP) mutation detection analysis presented in UNIT 7.4 is a very simple and highly sensitive mutation detection method. In this unit, the technology has been converted to a high-throughput screening method by performing the separation analysis in automated capillary electrophoresis instruments. Instructions describe the use of both single capillary instruments and capillary array instruments. Automated SSCP generates large amounts of data—thus, data analysis is clearly explained and detailed.
UNIT 7.13 One-Step Enzymatic Purification of PCR Products for Direct Sequencing (Jae Bum Kim and Seth Blackshaw, Harvard Medical School). The polymerase chain reaction (PCR) has become a powerful and widespread method for analyzing DNA. This unit will outline a quick (~30 min) and cost-effective method of enzymatically purifying PCR products for direct sequencing. The convenience and cost effectiveness of this protocol makes this approach ideal for large-scale sequencing projects such as serial analysis of gene expression (SAGE) and searching candidate genes for mutations.
UNIT 11.6 Serial Analysis of Gene Expression (SAGE; Seth Blackshaw, Jae B. Kim, Brad St. Croix, and Kornelia Polyak, Harvard Medical School, Boston MA). Serial analysis of gene expression (SAGE) is a powerful technique that allows comprehensive measurement of all mRNAs expressed in a sample of interest. The authors will present a reliable method of generating SAGE libraries from as little as 100,000 cells or one microgram of total RNA. Because SAGE analysis generates absolute rather than relative measurements of RNA abundance levels, it enables researchers to compare their data to those produced in other labs.
NEW CHAPTER !
Model Systems for the Analysis of Human Disease
This new chapter will offer users the background necessary to design studies of human diseases in animal systems. The emphasis will be on experimental objectives and strategic planning, and will identify potential applications, drawbacks, practical considerations, and resources needed to work with a core facility to set up a model.
The chapter will begin with an overview of the entire technology. This unit will come in a future supplement. However, we have initially published an overview on the use of mouse models for human disease, as described below. Other units will focus on Drosophila, Zebrafish, Xenopus, C. elegans, and yeast models.
UNIT 15.2 Use of Mouse Models for Human Disease (Christopher Semsarian, Howard Hughes Medical Institute and Harvard Medical School). Genetically manipulated mouse models facilitate the study of a variety of proteins involved in human disease. Such models have improved our understanding of structure-function relationships of proteins within specific organ systems, while introduction of defined mutations in specific genes in the mouse genome has enabled us to understand molecular, biochemical and cytological aspects of a variety of human diseases.
FORTHCOMING
UNIT 6.9 Accessing the Human Genome (Deanna Church, National Center for Biotechnology Information, Bethesda, MD). The vast amount of human data that has been generated over the last 15 months can be overwhelming. This unit will guide the user through the various web resources that attempt to assemble and annotate the human genome, including Ensembl, The Genome Browser, and NCBI Genome Resources.
Revision of UNIT 9.5, Molecular Analysis of Fragile X Syndrome (W. Ted Brown, The Institute for Basic Research, NY). The fragile X syndrome is the most common inherited cause of mental retardation. Diagnostic testing for the underlying triplet repeat mutation is important due to its prevalence and significance when detected. The mutation is due to expansion of a CGG repeat. The high CG content (~100%) makes the sequence particularly troublesome to amplify using PCR. The various methods in use with be compared and updated. Newer approaches will be detailed.
UNIT 9.13 Quantitation of X Inactivation Patterns (Eric Hoffman, Children's National Medical Center, Washington DC). The identification of X inactivation patterns in females is often key to many different lines of investigation, such as cell clonality (cancer), molecular diagnosis of X-linked disease, and recurrent pregnancy loss (cell lethal traits). Modern methods of quantitation of X inactivation patterns will be described using methylation-sensitive enzymatic digestion of genomic DNA, and detection of allele-specific PCR products on automated sequencers.