Raedle J, Brieger A, Trojan J, Hardt T, Herrmann G, Zeuzem S
Second Department of Internal Medicine, Johann Wolfgang Goethe University, Frankfurt/Main, Germany.
Mod Pathol. 1999 May;12(5):485-91.
Length alterations in short repetitive DNA sequences, termed microsatellite instability (MSI), are used as a diagnostic criterion of replication errors caused by various mutations in at least five mismatch repair genes. Therefore, MSI analysis is useful in clinical practice to identify patients with hereditary nonpolyposis colorectal cancer (HNPCC). MSI can be detected by amplification of microsatellite loci in DNA extracted from paraffin-embedded tumor and corresponding peritumoral specimens after numerous time consuming steps limiting the clinical utilities. Rapid microsatellite analysis, a efficient and rapid DNA extraction technique based on Triton X-100 preincubation, was compared with the conventional DNA extraction for HNPCC screening in colorectal tumor specimens from 12 patients. Five complex and two noncomplex (CA)n microsatellite loci were tested, with use of multicolor fluorescent analysis. MSI and loss of heterozygosity in colorectal tumor samples could equally be assessed with the two DNA preparation methods, whereas the number of initially unsuccessful DNA extractions from paraffin-embedded tissue specimens and overall duration for MSI analysis were significantly reduced when rapid microsatellite analysis was used. A replication error-positive phenotype was detected in 2 of 10 patients with a positive family history for colorectal cancer, and diagnosis of HNPCC was finally confirmed by detection of a specific germline mutation. The described rapid microsatellite analysis is less time consuming and more efficient, and, in general, it reduces the risk of contamination by limiting the number of steps required. Therefore, it might replace current DNA extraction procedures. Furthermore, techniques using fluorescent polymerase chain reaction and semiautomated DNA sequencer allow for precise, observer-independent, and rapid scoring in MSI and loss of heterozygosity assessment. A combination of our rapid DNA extraction method and the use of a highly specific microsatellite marker might improve replication error analysis in HNPCC screening.
短重复DNA序列的长度改变，即微卫星不稳定性（MSI），被用作由至少五个错配修复基因中的各种突变引起的复制错误的诊断标准。因此，MSI分析在临床实践中对于识别遗传性非息肉病性结直肠癌（HNPCC）患者很有用。在经过众多耗时步骤后，通过扩增从石蜡包埋肿瘤及相应肿瘤周围标本中提取的DNA中的微卫星位点，可以检测到MSI，但这限制了其临床应用。快速微卫星分析是一种基于Triton X-100预孵育的高效快速DNA提取技术，我们将其与传统DNA提取方法进行比较，用于对12例患者结直肠肿瘤标本进行HNPCC筛查。使用多色荧光分析检测了五个复杂和两个非复杂（CA）n微卫星位点。两种DNA制备方法均可同等评估结直肠肿瘤样本中的MSI和杂合性缺失，而使用快速微卫星分析时，从石蜡包埋组织标本中最初未成功提取DNA的数量以及MSI分析的总时长均显著减少。在10例有结直肠癌家族史阳性的患者中，有2例检测到复制错误阳性表型，最终通过检测特定的种系突变确诊为HNPCC。所描述的快速微卫星分析耗时更少、效率更高，并且一般来说，通过限制所需步骤数量降低了污染风险。因此，它可能会取代当前的DNA提取程序。此外，使用荧光聚合酶链反应和半自动DNA测序仪的技术能够在MSI和杂合性缺失评估中进行精确、独立于观察者的快速评分。我们的快速DNA提取方法与使用高度特异性微卫星标记相结合，可能会改善HNPCC筛查中的复制错误分析。
