Linkage Analysis and Positional Cloning

Linkage analysis can be used to pinpoint which chromosomal regions contain disease alleles by scanning the genome of family members, within affected pedigrees, for alleles that appear to be linked to the disease phenotype. Two loci are linked, if they are found on the same chromosomal segment and do not assort independently during meiosis (i.e., have a recombination frequency of less than 50%). Linkage analysis has been quite successful at determining loci that contribute moderate to large effects to disease predisposition, as is the case for monogenic and oligogenic disorders. However, this type of analysis has been generally unsuccessful in mapping loci involved in complex, polygenic disorders, for which each locus has only an incremental effect on the expression of the disease phenotype.

For an allele to be informative for linkage studies, it must be polymorphic, existing in more than one form within the population. Consequently, the power of this approach is limited by the number of affected family pedigrees that can be examined and the availability of informative polymorphic markers. Traditionally, restriction fragment length polymorphisms (RFLPs) and variable number of tandem repeats (VNTRs) have been used for linkage analysis. RFLPs rely on the differential location of restriction sites within DNA and are less polymorphic than VNTRs, which rely on the variability in length of minisatellite DNA. More recently, polymerase chain reaction (PCR) based genotypirig of DNA microsatellites and single nucleotide polymorphisms (SNPs) have been used in linkage analysis.

In the case of cystic fibrosis, extensive linkage analysis indicated that a single locus on chromosome 7, between bands q21 and q31, was linked to the disease allele (WHITEet al., 1985).Once this candidate region was identified, the DNA within this region was then scanned for genes that had the expression pattern or enzymatic activity expected for a candidate gene for cystic fibrosis. This method of gene cloning is called positional cloning. As a last confirmation step each candidate gene within the chromosomal region, identified by linkage analysis, must be screened for germline mutations that are only present in individuals with the disease phenotype

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