Boyle E B, Steinbuch M, Tekautz T, Gutman J R, Robison L L, Perentesis J P
Division of Pediatric Hematology, University of Minnesota Cancer Center, Minneapolis 55455, USA.
Cancer Epidemiol Biomarkers Prev. 1998 Dec;7(12):1127-31.
Archival slides are a potentially useful source of DNA for mutation analyses in large population-based studies. However, it is unknown whether specimen age or histological stains alter the accuracy of Taq polymerase or induce secondary mutations in sample DNA. To address this question, we evaluated five methods for extraction of genomic DNA from archival bone marrow slides of 17 leukemia patients and analyzed exons 1 and 2 of the N- and K-ras genes for the presence of mutations. Of the five methods, optimal DNA purification was achieved by boiling and phenol:chloroform extraction. N-and K-ras exons 1 and 2 were independently amplified using 35 cycles of PCR, and 6-12 clones for each exon were isolated and individually sequenced for each patient. Mutations were confirmed by repeat extraction, cloning, and sequencing. Sixteen of 17 patient samples were successfully amplified (94%), including slides up to 29 years old. Twelve slides had been stained with Wright-Giemsa, I stained with toluidine blue, and 4 were unstained. A total of 16 single-base mutations were identified of 33,840 nucleotides sequenced. No insertions or deletions were identified. Six of 16 single-base mutations were previously described activating mutations in codon 13 of N-ras exon 1. The 10 other mutations were in other regions of the N- and K-ras genes and were not reproduced after repeat extraction, cloning, and sequencing. The frequency of these other alterations was I of 3384 bp. This value is comparable with the inherent error frequency for Taq polymerase. Our findings suggest that high fidelity DNA amplification can be achieved using archival hematological slides as old as 29 years and can be reliably used in genetic analyses.
在大规模人群研究中，存档玻片是用于突变分析的潜在有用DNA来源。然而，尚不清楚样本保存时间或组织学染色是否会改变Taq聚合酶的准确性或在样本DNA中诱导二次突变。为了解决这个问题，我们评估了从17例白血病患者的存档骨髓玻片中提取基因组DNA的五种方法，并分析了N-和K-ras基因的第1和第2外显子是否存在突变。在这五种方法中，通过煮沸和苯酚：氯仿提取可实现最佳的DNA纯化。使用35个循环的PCR独立扩增N-和K-ras基因的第1和第2外显子，对每位患者的每个外显子分离6-12个克隆并分别测序。通过重复提取、克隆和测序确认突变。17例患者样本中有16例成功扩增（94%），包括保存长达29年的玻片。12张玻片用瑞氏-吉姆萨染色，1张用甲苯胺蓝染色，4张未染色。在测序的33,840个核苷酸中总共鉴定出16个单碱基突变。未鉴定到插入或缺失。16个单碱基突变中有6个是先前描述的N-ras基因第1外显子密码子13的激活突变。其他10个突变位于N-和K-ras基因的其他区域，在重复提取、克隆和测序后未重现。这些其他改变的频率为3384 bp中有1个。该值与Taq聚合酶的固有错误频率相当。我们的研究结果表明，使用保存长达29年的存档血液学玻片可以实现高保真DNA扩增，并可可靠地用于基因分析。
