Minisatellite "isoallele" discrimination in pseudohomozygotes by single molecule PCR and variant repeat mapping.
The D1S8 hypervariable minisatellite MS32 has a heterozygosity of 97.5% based on detectable differences in allele length using standard Southern blot analysis. It has previously been shown that the basic repeat unit is in itself variable and that this may be used to map the internal structure of an allele. This method has already been used to establish that alleles of the same length may have differing internal structures between nonrelated individuals. We now extend this approach to demonstrate that two apparently homozygous individuals are in fact heterozygotes. For each individual the two comigratory alleles were separated, without cloning, using single molecule dilution (SMD) of genomic DNA and recovery with PCR. Mapping of the variant repeat units revealed highly diverged internal structures and, for one individual, a size difference of one repeat unit (29 bp). SMD and PCR recovery provide an efficient system for separating comigratory alleles without prerequirement for knowledge of sequence differences.
Monckton
D G
DG
Department of Genetics, University of Leicester, United Kingdom.
Jeffreys
A J
AJ
D000483:Alleles; D001483:Base Sequence; D004276:DNA, Satellite / Q000235:genetics*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D004276:DNA, Satellite