Goodrow T L, Prahalada S R, Storer R D, Manam S V, Leander K R, Kraynak A R, van Zwieten M J, Nichols W W, Bradley M O
Merck, Sharp, and Dohme Research Laboratories, West Point, Pennsylvania.
Lab Invest. 1992 Apr;66(4):504-11.
The efficiency of detection of H- and K-ras mutations in 27 CD-1 mouse liver tumors by direct sequencing of polymerase chain reaction (PCR)-amplified DNA isolated from formalin-fixed and paraffin-embedded tissues was compared with that after assay by both NIH 3T3 transfection (followed by sequencing of amplified transformant DNA) and direct sequencing of PCR-amplified DNA isolated from frozen tumors. Some tumor samples were chosen for comparison because they contained ras mutations that were detected by either NIH 3T3 transfection or sequencing of PCR-amplified DNA derived from frozen tumors, but were not detected by both techniques. The efficiency of detecting K-ras mutations was similar for sequencing of amplified fragments derived from both paraffin-embedded tissues and from frozen tumors. However, these two techniques differed in their efficacy for detection of H-ras codon 61 mutations. Furthermore, this difference appeared to be mutation-specific: the sequencing of amplified products from paraffin-embedded tumor tissues allowed increased detection of CAA to AAA mutations but decreased detection of CAA to CTA mutations relative to sequencing of amplified fragments derived from frozen tumor DNA. Direct sequencing of PCR products from paraffin-embedded sections was more sensitive than NIH 3T3 transfection for detection of activated K-ras genes containing codon 13 mutations but less sensitive for detection of activated H-ras genes containing codon 61 mutations. In summary, direct sequencing of amplified DNA from either frozen tumors or formalin-fixed, paraffin-embedded tissues can be more sensitive than NIH 3T3 transfection for detection of codon 13-activated K-ras genes. However, it appears to be less sensitive than NIH 3T3 transfection for detection of certain activating H-ras mutations. Depending upon the questions being asked of the data, each of the methods can provide useful information about ras gene mutations in tumor samples. The apparent differences in sensitivities between the methods is not yet understood, but such differences should be considered in the analysis of data obtained when only one method is used.
通过对从福尔马林固定石蜡包埋组织中分离的聚合酶链反应（PCR）扩增DNA进行直接测序，检测27个CD-1小鼠肝肿瘤中H-和K-ras突变的效率，与通过NIH 3T3转染（随后对扩增的转化体DNA进行测序）以及对从冷冻肿瘤中分离的PCR扩增DNA进行直接测序后的检测效率进行了比较。选择一些肿瘤样本进行比较，因为它们含有通过NIH 3T3转染或从冷冻肿瘤衍生的PCR扩增DNA测序检测到的ras突变，但并非两种技术都能检测到。从石蜡包埋组织和冷冻肿瘤衍生的扩增片段测序检测K-ras突变的效率相似。然而，这两种技术在检测H-ras密码子61突变的效力上有所不同。此外，这种差异似乎是突变特异性的：相对于从冷冻肿瘤DNA衍生的扩增片段测序，石蜡包埋肿瘤组织扩增产物的测序允许增加CAA到AAA突变的检测，但减少CAA到CTA突变的检测。从石蜡包埋切片的PCR产物直接测序在检测含有密码子13突变的活化K-ras基因方面比NIH 3T3转染更敏感，但在检测含有密码子61突变的活化H-ras基因方面敏感性较低。总之，对于检测密码子13活化的K-ras基因，从冷冻肿瘤或福尔马林固定石蜡包埋组织的扩增DNA直接测序可能比NIH 3T3转染更敏感。然而，对于检测某些活化的H-ras突变，它似乎比NIH 3T3转染敏感性更低。根据对数据提出的问题，每种方法都可以提供有关肿瘤样本中ras基因突变的有用信息。这些方法之间敏感性的明显差异尚未明确，但在仅使用一种方法获得的数据的分析中应考虑这种差异。
