Turner Daniel J, Shendure Jay, Porreca Greg, Church George, Green Peter, Tyler-Smith Chris, Hurles Matthew E
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.
Nat Methods. 2006 Jun;3(6):439-45. doi: 10.1038/nmeth881.
Inversions are an important form of structural variation, but they are difficult to characterize, as their breakpoints often fall within inverted repeats. We have developed a method called 'haplotype fusion' in which an inversion breakpoint is genotyped by performing fusion PCR on single molecules of human genomic DNA. Fusing single-copy sequences bracketing an inversion breakpoint generates orientation-specific PCR products, exemplified by a genotyping assay for the int22 hemophilia A inversion on Xq28. Furthermore, we demonstrated that inversion events with breakpoints embedded within long (>100 kb) inverted repeats can be genotyped by haplotype-fusion PCR followed by bead-based single-molecule haplotyping on repeat-specific markers bracketing the inversion breakpoint. We illustrate this method by genotyping a Yp paracentric inversion sponsored by >300-kb-long inverted repeats. The generality of our methods to survey for, and genotype chromosomal inversions should help our understanding of the contribution of inversions to genomic variation, inherited diseases and cancer.
倒位是结构变异的一种重要形式，但由于其断点常常位于反向重复序列内，所以很难进行特征描述。我们开发了一种名为“单倍型融合”的方法，其中通过对人类基因组DNA的单分子进行融合PCR来对倒位断点进行基因分型。融合位于倒位断点两侧的单拷贝序列会产生方向特异性的PCR产物，以Xq28上int22血友病A倒位的基因分型检测为例。此外，我们证明，对于断点嵌入长（>100 kb）反向重复序列内的倒位事件，可通过单倍型融合PCR，随后对位于倒位断点两侧的重复特异性标记进行基于微珠的单分子单倍型分型来进行基因分型。我们通过对由>300 kb长的反向重复序列引发的Yp臂间倒位进行基因分型来说明这一方法。我们用于检测和对染色体倒位进行基因分型的方法具有通用性，这应该有助于我们理解倒位对基因组变异、遗传性疾病和癌症的影响。
