Nairz Knud, Stocker Hugo, Schindelholz Benno, Hafen Ernst
Zoologisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10575-80. doi: 10.1073/pnas.162136299. Epub 2002 Jul 29.
With the availability of complete genome sequences, new rapid and reliable strategies for positional cloning become possible. Single-nucleotide polymorphisms (SNPs) permit the mapping of mutations at a resolution not amenable to classical genetics. Here we describe a SNP mapping procedure that relies on resolving polymorphisms by denaturing HPLC without the necessity of determining the nature of the SNPs. With the example of mapping mutations to the Drosophila nicastrin locus, we discuss the benefits of this method, evaluate the frequency of closely linked and potentially misleading second site mutations, and demonstrate the use of denaturing high-performance liquid chromatography to identify mutations in the candidate genes and to fine-map chromosomal breakpoints. Furthermore, we show that recombination events are not uniformly dispersed over the investigated region but rather occur at hot spots.
随着全基因组序列的可得性，新的快速且可靠的定位克隆策略成为可能。单核苷酸多态性（SNP）使得以经典遗传学无法达到的分辨率来定位突变成为可能。在此，我们描述一种SNP定位程序，该程序依靠变性高效液相色谱法（HPLC）解析多态性，而无需确定SNP的性质。以将突变定位到果蝇尼卡斯特林基因座为例，我们讨论了该方法的优点，评估紧密连锁且可能产生误导的第二位点突变的频率，并展示如何使用变性高效液相色谱法来鉴定候选基因中的突变以及对染色体断点进行精细定位。此外，我们表明重组事件并非均匀分布在所研究的区域，而是发生在热点区域。
