Skip to main content

Advertisement

Figure 1 | BMC Genetics

Figure 1

From: Robust physical methods that enrich genomic regions identical by descent for linkage studies: confirmation of a locus for osteogenesis imperfecta

Figure 1

Physical IBD enrichment process. Genomic DNAs (gDNA) are isolated from two related, afflicted individuals and digested with a restriction enzyme that leaves Exonuclease III-resistant ends and generates fragments of about 4 Kb. DNA fragments are modified (diamonds or ovals) to permit discrimination between the individuals, for example by presence or absence of methylation at GATC sequences. The fragments are mixed, denatured and renatured under conditions to favour unique copy reannealing. Non-annealed strands and hybrids of strands from the same individual are eliminated. Reannealed DNA fragments with one strand from each individual are called heterohybrids, which may be perfectly paired due to inheritance of both strands from the same ancestral sequence or mismatched due to variation between different ancestral sequences. Mismatched heterohybrid fragments are removed by LSHase, a nucleolytic cocktail of MutL, MutS and MutH, and subsequent digestion by Exonuclease III. The resulting IBD-enriched DNA is generically amplified, labelled and mapped by two-colour hybridization to genomic topographic arrays, using the reannealed DNA as reference. The process is repeated for other afflicted pairs in the same family and in additional families. Variations include use of oligonucleotides as discrimination tags, reducing the number of steps by combining similar intermediate purification procedures, and eventually mapping IBD regions by high throughput redundant sequencing, with or without amplification.

Back to article page