Genetic mapping using fluorescent quantification of allele frequencies in pooled DNA loaded by solid support.
An efficient and labour-saving method for fragment analysis in linkage studies using biotinylated primers and streptavidin-coated combs is presented. The level of streptavidin attached to the combs was used to control the amount of immobilised material. Thus, the need for titration of PCR products to fit the dynamic range of the sequencer was reduced. The method was used to investigate the possibility of quantitating allele frequencies in pools of DNA from family members with the autosomal dominant eye disorder Best's macular dystrophy. The method allowed the detection of one unique allele in a background of 39 other alleles. Using independent datasets, it was further found that the method was able to detect distorted allele frequencies in affected individuals of one family as compared to reference individuals, for markers located more than 30 cM from the disease locus. It was found that this procedure is a powerful alternative to conventional linkage analysis and the method may prove useful in a genome scan for genes involved in complex disorders.
Graff
C
C
Department of Clinical Genetics, University Hospital, Uppsala, Sweden.
Persson
A
A
Ulfendahl
P J
PJ
Wahlberg
J
J
Wadelius
C
C
D000483:Alleles; D002874:Chromosome Mapping / Q000379:methods*; D004247:DNA / Q000032:analysis*; D008040:Genetic Linkage; D005819:Genetic Markers; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics; D018895:Microsatellite Repeats / Q000235:genetics*; D016133:Polymerase Chain Reaction
D005819:Genetic Markers; D004247:DNA