Department of Haematology, Royal Infirmary of Edinburgh, Little France Crescent, Edinburgh EH16 4SA, UK.
BMC Genomics. 2010 Apr 6;11:223. doi: 10.1186/1471-2164-11-223.
One of the major obstacles to the exploitation of genetic variation in human medicine, veterinary medicine, and animal breeding is the difficulty in defining haplotypes in unrelated individuals.
We have developed a Multiplex Double Amplification Refractory Mutation System combined with Solid Phase PCR on Fluorescently labelled beads. The process is inherently amenable to automation. It provides a high degree of internal Quality Control, as each PCR product is represented in duplicate on the bead array, and each SNP is tested against multiple partners. This technique can resolve very complex genotypes into their constituent haplotypes; it defined all the alleles at 60 SNP in exon 2 of the ovine DRB1 MHC locus in a sample of 109 rams. These 60 SNP formed 33 DRB1 exon 2 alleles; two of which had not been previously identified; although both of them have been independently confirmed.
This technique has the same resolution as allele specific sequencing. Sequencing has the advantage of identifying novel polymorphic sites but where all SNP sites have been identified this novel procedure can resolve all alleles and haplotypes and identify novel combinations of polymorphisms. This method is similar in price to direct sequencing and provides a low cost system for direct haplotyping of extended DNA sequences.
在人类医学、兽医和动物育种中利用遗传变异的主要障碍之一是难以在非相关个体中定义单倍型。
我们开发了一种多重双扩增阻断突变系统，结合荧光标记珠上的固相 PCR。该过程本质上易于自动化。它提供了高度的内部质量控制，因为每个 PCR 产物在珠阵列上重复表示两次，并且每个 SNP 都与多个伙伴进行测试。该技术可以将非常复杂的基因型解析为其组成的单倍型；它在 109 只公羊的样本中确定了绵羊 DRB1 MHC 基因座外显子 2 中 60 个 SNP 的所有等位基因。这 60 个 SNP 形成了 33 个 DRB1 外显子 2 等位基因；其中两个以前没有被识别；尽管它们都已经被独立证实。
该技术与等位基因特异性测序具有相同的分辨率。测序具有识别新多态性位点的优势，但在所有 SNP 位点都已被识别的情况下，该新程序可以解析所有等位基因和单倍型，并识别新的多态性组合。该方法与直接测序的价格相似，为直接对扩展 DNA 序列进行单倍型分析提供了一种低成本系统。
