Horikoshi T, Lenz H J, Danenberg K, Koch O M, Bertino J R, Danenberg P V
Kenneth Norris Jr Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles 90033.
Leuk Res. 1994 Sep;18(9):693-702. doi: 10.1016/0145-2126(94)90069-8.
By combining allele-specific PCR amplification and a PCR-based quantitation approach, a method has been developed to estimate the mutated K-ras gene content in the blood of AML patients as a percentage of total K-ras. One PCR primer set was designed not to discriminate between mutant K-ras and wild-type K-ras and thus amplified the total K-ras gene. The other PCR primer set was designed to be allele-specific for K-ras gene containing a G to C mutation at codon 12. This primer set could discriminate the mutant and wild-type genes when the proportion of the mutated sequence was 0.2% of the total K-ras gene. To test the method on biological specimens, genomic DNA samples were analyzed from the peripheral blood of a patient who had secondary AML with the same codon 12 K-ras mutation. Two samples taken from this patient 2 months apart during follow-up had myeloblast cell contents of 67 and 80%. However, the percentage of mutated K-ras was 50% in both samples, suggesting that this patient may be inherently heterozygotic in this particular mutation. This ratio of mutated to normal K-ras in the patient's cells was confirmed by RNA-SSCP analysis and RNA sequencing. This quantitation method can provide a sensitive and specific estimation of the content of mutated K-ras alleles in patient samples.
通过结合等位基因特异性PCR扩增和基于PCR的定量方法，已开发出一种方法来估计急性髓系白血病（AML）患者血液中突变型K-ras基因含量占总K-ras基因的百分比。设计了一组PCR引物，使其不区分突变型K-ras和野生型K-ras，从而扩增总K-ras基因。另一组PCR引物被设计为对密码子12处含有G到C突变的K-ras基因具有等位基因特异性。当突变序列的比例占总K-ras基因的0.2%时，该引物组能够区分突变型和野生型基因。为了在生物样本上测试该方法，对一名患有继发性AML且密码子12处存在相同K-ras突变的患者外周血的基因组DNA样本进行了分析。在随访期间，从该患者身上相隔2个月采集的两份样本中，成髓细胞含量分别为67%和80%。然而，两份样本中突变型K-ras的百分比均为50%，这表明该患者在这一特定突变中可能固有地为杂合子。通过RNA-SSCP分析和RNA测序证实了患者细胞中突变型与正常K-ras的这一比例。这种定量方法可以对患者样本中突变型K-ras等位基因的含量进行灵敏且特异的估计。
