Hansen Helen M, Wiemels Joseph L, Wrensch Margaret, Wiencke John K
Department of Neurological Surgery, University of California, Suite AC-34, 1 Irving Street, San Francisco, CA 94143-0441, USA.
Cancer Epidemiol Biomarkers Prev. 2007 Aug;16(8):1686-90. doi: 10.1158/1055-9965.EPI-06-1024.
Whole genome amplification (WGA) permits genotyping DNA samples of limited quantity, expanding the number of samples available for genetic epidemiology studies. WGA, however, produces various nucleic acid side products that may interfere with accurate DNA quantification and further analysis. Although quality control of whole genome amplified DNA (wgaDNA) before genotyping is essential to prevent failed or poor genotyping results, little information is available to choose the best method for wgaDNA quantification. Therefore, we quantified wgaDNA from 54 buccal or poor quality blood samples by four methods: UV absorbance, PicoGreen fluorescence calibrated with lambda bacteriophage or calf thymus DNA, and absolute quantification by real-time PCR amplification of human-specific Alu Yd6. We then genotyped these wgaDNA samples and paired high-quality genomic DNA samples on a custom 384-plex Illumina Golden Gate Panel. Of the 54 paired samples, 39 gave high concordance (>99%), whereas 7 had moderate concordance (>90-99%) and 8 had poor concordance (</=90%) of wgaDNA and genomic DNA genotyping results. Quantitative PCR of Alu was the only wgaDNA quantification method to distinguish wgaDNA samples that gave high, moderate, or low concordance results (i.e., wgaDNA quantities in the high, moderate, and poor concordance groups ranged at 4.14-118.32, 0.29-2.19, and 0.01-0.27 ng/microL, respectively). Human-specific quantitative PCR is a highly useful guide for determining the suitability of wgaDNA before high-throughput single-nucleotide polymorphism analysis.
全基因组扩增（WGA）可对少量DNA样本进行基因分型，从而增加了可用于遗传流行病学研究的样本数量。然而，WGA会产生各种核酸副产物，这些副产物可能会干扰DNA的准确定量和进一步分析。尽管在基因分型前对全基因组扩增DNA（wgaDNA）进行质量控制对于防止基因分型结果失败或不佳至关重要，但关于选择最佳wgaDNA定量方法的信息却很少。因此，我们采用四种方法对来自54份口腔拭子样本或质量较差的血液样本的wgaDNA进行了定量：紫外吸收法、用λ噬菌体或小牛胸腺DNA校准的PicoGreen荧光法，以及通过人类特异性Alu Yd6的实时PCR扩增进行绝对定量。然后，我们在定制的384重Illumina Golden Gate Panel上对这些wgaDNA样本和配对的高质量基因组DNA样本进行了基因分型。在这54对样本中，39对具有高度一致性（>99%），而7对具有中度一致性（>90 - 99%），8对wgaDNA和基因组DNA基因分型结果的一致性较差（≤90%）。Alu的定量PCR是唯一一种能够区分产生高、中、低一致性结果的wgaDNA样本的定量方法（即，高、中、低一致性组中的wgaDNA量分别在4.14 - 118.32、0.29 - 2.19和0.01 - 0.27 ng/μL范围内）。在进行高通量单核苷酸多态性分析之前，人类特异性定量PCR是确定wgaDNA适用性的非常有用的指导方法。
