Spiegelman J I, Mindrinos M N, Oefner P J
Stanford Genome Technology Center, Palo Alto, CA, USA.
Biotechniques. 2000 Nov;29(5):1084-90, 1092. doi: 10.2144/00295rr04.
Genetic maps based on biallelic single-nucleotide polymorphisms amenable to microarray-based genotyping have significantly accelerated the mapping of mono- and multigenic traits in model organisms such as Saccharomyces cerevisiae and Arabidopsis thaliana. This advance needs to be matched by highly accurate, inexpensive and robust methodology for fine-structure mapping of the candidate region(s) and the eventual identification of the causative mutation(s). To establish the usefulness of denaturing high-performance liquid chromatography (DHPLC) for those purposes, we have amplified 476 fragments from two A. thaliana ecotypes with an average length of 563 bp covering various candidate regions on chromosomes 1, 2 and 4. Parallel analysis by DHPLC and dye terminator sequencing showed that DHPLC detected 165 out of 166 polymorphic fragments with only four false positives, amounting to a sensitivity, specificity and accuracy of 99.4%, 98.7% and 99%, respectively. It proved beneficial to analyze the fragments not only at the highest but also at the lower temperatures recommended by the algorithm freely available at http:¿insertion.stanford.edu/melt.html.
基于双等位基因单核苷酸多态性且适用于基于微阵列基因分型的遗传图谱，显著加速了酿酒酵母和拟南芥等模式生物中单基因和多基因性状的图谱绘制。这一进展需要与用于候选区域精细结构图谱绘制以及最终鉴定致病突变的高度准确、廉价且稳健的方法相匹配。为了确定变性高效液相色谱（DHPLC）用于这些目的的实用性，我们从两个拟南芥生态型中扩增了476个片段，平均长度为563 bp，覆盖了第1、2和4号染色体上的各个候选区域。通过DHPLC和染料终止子测序进行的平行分析表明，DHPLC在166个多态性片段中检测到165个，仅有4个假阳性，灵敏度、特异性和准确率分别为99.4%、98.7%和99%。结果证明，不仅在由http://insertion.stanford.edu/melt.html免费提供的算法推荐的最高温度下，而且在较低温度下分析片段都是有益的。
