Positional cloning, genomics

Comparative (Zoo-FISH) genome mapping and positional cloning of evolutionary chromosome breakpoints in important mammal and vertebral genomes... 

With completion of the human genome project, the functional and positional cloning of Mendelian traits and diseases can become fairly routine (2). Yet, considerable work still lies ahead even with Mendelian diseases document worldwide variation in mutations, identify the effects of modifier genes on heterogeneity with respect to age of onset and severity, obtain accurate measurements of the penetrance values of different allelic mutations, study the effects of environmental factors on disease expression, and develop appropriate therapies. 

By this approach, positive clones are expected to arise from overexpression of wild-type genes conferring resistance or from frans-dominant mutations giving a mutant (resistant) phenotype (14). To identify loss-of-function recessive mutations the method should be reversed P-RES resistant clones should be transformed with a genomic library constructed with the DNA of the wild-type HB101, and clones with restored peptide susceptibility analyzed. 

To reduce false-positive clones, mRNA should be prepared free from rRNA and genomic DNA as possible. We purify and concentrate mRNA by QuickPrep Micro mRNA purification Kit (Pharmacia) using glycogen as a carrier. Then, contaminated gemonic DNA is digested with RNase-free DNase I (Gibco-BRL). 

Because of the sequence complexity of genomic DNA, isolation of false positive clones by blot hybridization is a significant problem when the stringency of the washing conditions is very low (wash temperature <42°C). 

Characterize retrotransposons. Once a positive clone has been identified, it can be used as a probe to isolate full-length copies of the element from a genomic library. These clones can also be characterized by phylogenetic screening. 

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