Mátyás Gábor, Giunta Cecilia, Steinmann Beat, Hossle Johann Peter, Hellwig Robert
University Children's Hospital, Division of Metabolism and Molecular Pediatrics, Zurich, Switzerland.
Hum Mutat. 2002 Jan;19(1):58-68. doi: 10.1002/humu.10013.
We present a novel method for accurate quantification of single nucleotide polymorphism (SNP) variants in transcripts and pooled DNAs in a one-tube reaction. Our approach is based on single- nucleotide primer extension (SNuPE) and laser-induced fluorescence capillary electrophoresis (LIF-CE), and takes advantage of distinct mobilities of SNuPE products with different nucleotides incorporated at their 3' ends. The method, called SNuPE-ONCE, was tested on two polymorphisms and five mutations that comprised the three most frequent ( approximately 70%) nucleotide changes in the human genome (C/T, A/G, and A/T). The usefulness of the method was demonstrated by analyzing nonsense-mediated mRNA instability in fibroblasts. Our data show 1) that the method provides highly reproducible relative allele frequencies (SD<0.017) with a good accuracy (e.g. for heterozygotes 0.500 +/- 0.036, P = 0.01), depending on the sequence and the proportion of the SNP variants in the sample, and 2) that relative allele frequencies as low as 1% can be detected quantitatively and unambiguously. Our assay relies on a CE instrument available in many laboratories and offers a useful method for quantitative SNP genotyping as well as for a variety of expression studies.
我们提出了一种新颖的方法，可在单管反应中对转录本和混合DNA中的单核苷酸多态性（SNP）变体进行准确定量。我们的方法基于单核苷酸引物延伸（SNuPE）和激光诱导荧光毛细管电泳（LIF-CE），并利用了在其3'末端掺入不同核苷酸的SNuPE产物的不同迁移率。该方法称为SNuPE-ONCE，已针对两种多态性和五种突变进行了测试，这些突变构成了人类基因组中三种最常见（约70%）的核苷酸变化（C/T、A/G和A/T）。通过分析成纤维细胞中无义介导的mRNA不稳定性，证明了该方法的实用性。我们的数据表明：1）该方法根据样本中SNP变体的序列和比例，提供了高度可重复的相对等位基因频率（SD<0.017），且准确性良好（例如，杂合子为0.500±0.036，P = 0.01）；2）可以定量且明确地检测低至1%的相对等位基因频率。我们的检测方法依赖于许多实验室都有的CE仪器，并为定量SNP基因分型以及各种表达研究提供了一种有用的方法。
