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Isolation of Coding Sequences from Genomic Regions Using Direct Selection

The rate-limiting steps in the positional cloning of disease genes are the identification of discrete genes within a large genomic region and the analysis of mutations in these genes. Direct selection is an expression-based gene identification technique that can rapidly identify cDN ...

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Isolation of cDNAs Using the YAC Hybridization Screen Method

There are currently a number of techniques available for the isolation of novel coding sequences from cloned genomic fragments. The most recent methodologies include exon trapping (1), direct selection (2,3), and screening of cDNA libraries with whole radiolabeled yeast artificial c ...

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Chemical Crosslinking Subtraction (CCLS)

Since the advent of polymerase chain reaction (PCR), it has proven possible to clone complete genes, or parts of these, without having to construct the usually obligatory cDNA library. PCR cloning, however, is not without its own problems and limitations, such as the fidelity of the cloned sequence ...

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Detection and Isolation of Differentially Expressed Genes by Differential Display

Temporal and spatial expression of the 100,000 different genes in the genome of a mammal is a highly regulated process that determines the normal development of an organism. This assumption is based on the fact that only a fraction of these genes, in the range of 10,000–15,000, are expressed in any cell ty ...

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Gene Mapping and Isolation: Access to Databases

If two variants in a population are related by simple Mendelian inheritance, then in principle, it is possible to isolate a gene in which the sequence differences will explain the nature of the variants. In practice, this is a daunting task in a genome the size of human (3000 Mb) and may take many years to achieve. ...

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Software for Genetic Linkage Analysis: An Update

Reviews describing the software available to support genetic linkage analysis have appeared intermittently over the last few years (1,1a). This period has been associated with several significant influences that have made it timely to reconsider the available programs and related ...

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Gene Mapping Goes from FISH to Surfing the Net

The arrival of the second millennium will usher in unsurpassed information and knowledge of our genetic constitution, and will promise to revolutionize basic research and molecular medicine. The road toward a complete understanding of our genetic makeup is largely the fruit of the Human G ...

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Exon Detection by Similarity Searches

Other chapters of this volume have presented the various experimental methods (mainly exon trapping and recombination-based and hybridization-based approaches) used for the identification of transcribed sequences within cloned genomic fragments. None of those methods re ...

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Linkage Analysis of Genetic Disorders

Genetic disorders follow a classic Mendelian dominant or recessive single-locus pattern of inheritance or a complex genetic pattern (multiple genes and environmental influences). In general, the complexity arises when the simple correspondence between genotype and phenoty ...

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Gene Ordering and Localization by Linkage Analysis

The essence of linkage analysis is a deviation from the Mendelian principle of independent, or random, assortment of gene pairs when transmitted from generation to generation. Two genes are said to be completely linked (see Section 4.1. for definitions of genetic terms) when there is no recombi ...

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Gene Mapping Using Somatic Cell Hybrids

Human-rodent somatic cell hybrids have proven to be a useful tool for mapping expressed gene products (1,2) and, in particular, DNA sequences. The accuracy of the method is dependent on the identification of a species difference between the human and rodent species. This chapter details metho ...

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Probe Ordering and Distancing by FISH

In situ hybridization (ISH) has proven to be a powerful methodological approach to visualize specific nucleic acid sequences directly within the morphological context of the cell. The method involves hybridization of labeled probes to denatured target chromatin that has been fixed on ...

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Physical Mapping by Pulsed-Field Gel Electrophoresis

During the past few years, there has been intense activity in physically mapping the genomes of a variety of organisms, including human, mouse, Drosophila, Caenorhabditis elegans, Arabidopsis thaliana, and yeast. The considerable advances made in physically mapping these genomes has ...

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Construction and Use of Cosmid Contigs

The application of cosmids to positional cloning strategies has changed over the last several years (1). Initially, the construction of genome physical maps, for example, in Caenorhadbitis elegans and Arabidopsis (2,3), was attempted using cosmid clones randomly selected and analyz ...

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Construction and Use of YAC Contigs in Disease Regions

The identification of genes by positional cloning has revolutionized the molecular analysis of disease. Disease-causing genes can now be isolated with no knowledge of the biological function of the molecule involved, relying only on genetic inheritance or chromosomal perturbat ...

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Reduced Representation Methods for Subgenomic Enrichment and Next-Generation Sequencing

Several methods have been developed to enrich DNA for subsets of the genome prior to next-generation sequencing. These front-end enrichment strategies provide powerful and cost-effective tools for researchers interested in collecting large-scale genomic sequence data. In this ...

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Genomic Libraries: II. Subcloning, Sequencing, and Assembling Large-Insert Genomic DNA Clones

Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger me ...

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Genomic Libraries: I. Construction and Screening of Fosmid Genomic Libraries

Large insert genome libraries have been a core resource required to sequence genomes, analyze haplotypes, and aid gene discovery. While next generation sequencing technologies are revolutionizing the field of genomics, traditional genome libraries will still be required for acc ...

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Chromosome Analysis in Invertebrates and Vertebrates

The revolution in molecular techniques over the last 30 years detracted from many traditional cytological techniques for examining basic biological problems. One of these casualties is the preparation of karyotypes and analysis of chromosomal structure, behaviour, and variat ...

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Genome Size Determination Using Flow Cytometry of Propidium Iodide-Stained Nuclei

With the rapid expansion of whole-genome sequencing and other genomic studies in nonmodel �organisms, there is a growing demand for robust and user-friendly methods for estimating eukaryotic genome sizes across a broad range of taxa. Propidium iodide (PI) staining with flow cytometry is a ...

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