MALDI-TOF MS and TaqMan® assisted SNP genotyping of DNA isolated from formalin-fixed and paraffin-embedded tissues (FFPET)
MALDI-TOF MS and TaqMan® assisted SNP genotyping of DNA isolated from formalin-fixed and paraffin-embedded tissues (FFPET)
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DOI：
10.1002/humu.20141
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发表时间：
2005-01-01
期刊：
影响因子：
3.9
通讯作者：
Brauch, H
中科院分区：
文献类型：
--
作者：
Jaremko, M;Justenhoven, C;Brauch, H
来自存档临床样本的福尔马林固定石蜡包埋组织(FFPET)为大规模分子遗传学研究提供了宝贵的来源。需要患者长期临床随访数据的药物遗传学研究可能特别受益于FFPET分析。基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)和基于TaqMan(C)(Thermus Aquaticus聚合酶)的方法已成为标准的基因分型方法。然而，没有关于MALDI-TOF MS对低质量DNA进行基因分型的数据，因为它通常是从FFPET获得的，而TaqMan基因分型的数据也是有限的。我们从274例FFPET样本(229例乳腺癌患者和45例乳腺良性疾病患者)中提取了构成DNA，并对10个基因中的15个多态座位进行了基因分型。用MALDI-TOF MS对9个SNP进行了分型，用TaqMan方法对6个SNP进行了分型。我们建立了所有FFPET、1990年前准备的FFPET和1990年后准备的FFPET的等位基因分配成功率。MALDI-TOF MS和TaqMan基因分型的成功率分别为70.9%~99.6%(平均91.8%)和82.3%~97.7%(平均91.0%)。1990年前和1990年后制备的FFP ET的基因分型性能没有显著差异。除1个外，其余各基因频率均符合Hardy-Weinberg平衡。此外，在德国乳腺癌人群和其他高加索人群中观察到的基因频率与此相匹配。我们的研究首次表明，MALDI-TOF MS和TaqMan基因分型程序提供了可靠的数据，因此适用于需要大规模FFPET基因分型的研究。(C)2005年Wiley，Liss，Inc.
Formalin-fixed paraffin, embedded tissues (FFPET) from archived clinical samples provide an invaluable source for large-scale molecular genetic studies. Pharmacogenetic investigations that require long,term clinical follow-up data of patients may particularly benefit from FFPET analysis. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and TaqMan(C)-based (Thermus aquaticus polymerase) methodologies have become standard genotyping procedures. However, no data are available on the applicability of MALDI-TOF MS to the genotyping of low quality DNA, as it is usually obtained from FFPET, and data from TaqMan genotyping are limited. We isolated constitutional DNA from 274 FFPET samples (229 patients with breast cancer and 45 patients with benign breast diseases) and genotyped 15 polymorphic loci in 10 genes. Nine SNPs were genotyped by MALDI-TOF MS, and six were genotyped by the TaqMan methodology. We established rates for successful allele assignment for all FFPET, for FFPET prepared prior to 1990, and for FFPET prepared post,1990. Both methodologies showed high success rates ranging between 70.9 and 99.6% (mean: 91.8%) for MALDI-TOF MS and between 82.3 and 97.7% (mean: 91.0%) for TaqMan genotyping. No significant differences in genotyping performances for FFPET prepared prior to 1990 or post-1990 were observed. With the exception of one, all other genotype frequencies were in Hardy-Weinberg equilibrium. Furthermore, genotype frequencies matched those observed in a German breast cancer population and other Caucasian populations. Our study shows for the first time that MALDI-TOF MS and TaqMan genotyping procedures provide reliable data, and are therefore applicable in studies that require large scale FFPET genotyping. (C) 2005 Wiley,Liss, Inc.