Martínez-Arias Rosa, Bertranpetit Jaume, Comas David
Unitat de Biologia Evolutiva, Facultat de Ciències de la Salut i de la Vida, Universitat Pompeu Fabra, Doctor Aiguader 80, 08003 Barcelona, Spain.
DNA Seq. 2002 Feb;13(1):9-13. doi: 10.1080/10425170290019847.
Variation analyses in the human genome at the sequence level, especially human genetic population analysis and genetic epidemiology, are hampered by the difficulty to ascertain haplotypes on autosomal regions. We have designed a new methodological approach to obtain autosomal haploid sequences from diploid organisms. First, genotypes are unambiguously determined through long-range PCR and diploid DNA sequencing. Second, cloning the whole PCR-amplified segment and sequencing a single clone for those fragments that presented a heterozygous position discern the allelic phase. The second allele is deduced from the genotype, and the phase reconfirmed by sequencing a second clone. A hundred human chromosomes were analysed for a 5.4 kb encompassing the glucocerebrosidase pseudogene on human chromosome 1. Haplotypes were unambiguously ascertained for all samples. The manner to combine the used techniques makes this approach a novelty. Haploid sequences from diploid organisms are obtained in a less time consuming and more accurate manner than in other used procedures.
在序列水平上对人类基因组进行变异分析，尤其是人类遗传群体分析和遗传流行病学研究，因难以确定常染色体区域的单倍型而受到阻碍。我们设计了一种新的方法来从二倍体生物中获取常染色体单倍体序列。首先，通过长距离聚合酶链反应（PCR）和二倍体DNA测序明确确定基因型。其次，克隆整个PCR扩增片段，并对那些呈现杂合位置的片段测序单个克隆以辨别等位基因相位。从基因型推断出第二个等位基因，并通过对第二个克隆测序来再次确认相位。对包含人类1号染色体上葡萄糖脑苷脂酶假基因的5.4 kb区域的100条人类染色体进行了分析。所有样本的单倍型都得到了明确确定。所使用技术的组合方式使这种方法具有新颖性。与其他已使用的程序相比，以这种方式从二倍体生物中获取单倍体序列耗时更少且更准确。
