Inversions frequently result from non allelic homologous recombination between long (>10kb) inverted repeats, making them difficult to detect with long PCR or RFLP analysis. We have developed PCR-based single molecule haplotyping methods that enable both surveys for novel inversion variants, and population-scale genotyping of known inversions (Turner et al 2006).
The inversion genotype of an individual can be deduced from their genomic DNA if single molecule haplotyping is performed on the DNA fragments that span the inversion breakpoints. Fusion PCR (Yon and Fried 1989) allows sequences at either side of an inversion breakpoint to be joined together, and performing the reaction in an oil : water emulsion (Ghadessy et al 2001) ensures that the DNA templates are single molecules in the overwhelming majority of cases.
In practice, the haplotyping method used depends on the length of the inverted repeat relative to the size of DNA fragments in the genomic DNA preparation. If the repeat is shorter than the template fragment lengths (around 100kb), single copy sequences on either side of an inverted repeat can be joined and the inversion genotyped by generating fused products of different sizes for the two orientations.
For longer repeats, paralog-specific nucleotides are analysed in a magnetic bead-based version of the assay.