Positional cloning by exon trapping and cDNA selection

Auteurs

• Bernhard Korn

Paramètres

• Biologie
• Sciences naturelles

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Tremendous strides in decoding the human genome have occurred over the last decade, with advanced techniques significantly expanding the identification of genes and sequenced DNA. Despite these advancements, earlier gene isolation and cloning methods remain essential for constructing dense transcript maps to isolate coding sequences and disease-associated genes within specific chromosomal regions. This guide explores two powerful methods for locating gene coding regions by isolating gene fragments from individual clones or pools of genomic clones. It details the exon-trapping and cDNA selection processes step by step, covering everything from isolating genomic templates and nuclear splicing to verifying generated clones and analyzing exon libraries. Key procedures include descriptions of exon-trapping systems and vectors, exon amplification protocols, evaluation of exon libraries through PCR testing, and isolation of genomic templates. Additionally, it outlines cDNA selection experiment protocols, including cDNA screening and hybridization, as well as clone analysis. This resource serves as an invaluable tool for researchers, scientists, and graduate students, providing a critical approach to isolating specific disease genes within targeted genomic areas.

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• Année de publication: 1999