Major strategies:
Functional cloning - Using information about the function of a known
protein that could be involved in a genetic disease. This approach has
very limited application.
Candidate gene approach - Using information about the gene's possible function, homology, and expression pattern, but without any knowledge of its chromosomal location.
Positional cloning - Using only information about the gene's approximate chromosomal
location obtained from gene mapping.
Positional candidate approach - Using information from map position and the gene's possible function, homology, and expression pattern. This approach has been
quite successful and will dominate other strategies.
Between 1986 and 1995, more than 50 human disease genes were discovered by
pure positional cloning. A labor intensive and time consuming method
called chromosome
walking (see Alberts
et al.) is often involved in this approach. It took ten years for a
large group of researchers to clone the Huntington
disease gene. Cloning of the cystic
fibrosis gene took four years, which was facilitated by a special
technique called chromosome jumping (see Griffiths
et al.). In the future, as the entire human genome sequence is known, there is no
need to use chromosome walking (or jumping) to identify disease genes.
Book section:
Identifying human disease genes - From Human Molecular Genetics, 1999, by T. Strachan and A. P. Read.
Mutation detection methods
To confirm a candidate gene, it is often necessary to screen mutations from patient's
genes. In addition to direct DNA sequencing, the following methods have also been used.
SSCP (single strand conformational polymorphism analysis)
DGGE (Denaturing Gradient Gel Electrophoresis)
DHPLC (Denaturing High-Performance Liquid Chromatography
CCM (Chemical Cleavage of Mismatches)
EMC (Enzyme Mismatch Cleavage)
Heteroduplex analysis
DNA microarrays
Review articles:
Enabling
Large-Scale Pharmacogenetic Studies by High-Throughput Mutation
Detection and Genotyping Technologies - Clinical Chemistry, 2001
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