Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
Clin Biochem. 2010 Feb;43(3):296-301. doi: 10.1016/j.clinbiochem.2009.10.007. Epub 2009 Oct 29.
Mutations of all three RAS genes, N-, H-, and KRAS, are identified mainly in codons 12, 13, and 61 of exons 2 and 3 in human cancers.
DNA samples were isolated from 58 oral cancer and 106 colorectal cancer patients. Multiplex amplification of codons 12 and 13 of exon 2 and codon 61 of exon 3 of three RAS genes using two pairs of universal primers for exons 2 and 3 was performed in a single tube. The products were cleaned and split in three tubes. Each was subjected for primer extension using seven different-sized RAS primers for different RAS gene separately to detect base changes in codons 12, 13, and 61 of each RAS gene.
We compared the results with that from direct sequencing for detecting N-, H-, and KRAS mutations in 58 oral cancers and 106 colorectal cancers. The two methods yield identical results, but our method is superior to direct sequencing in terms the amount of work and time required.
We presented a rapid method to detect codons 12, 13, and 61 mutations of N-, H-, and KRAS genes in human cancers.
在人类癌症中，N-、H-和 KRAS 三个 RAS 基因的突变主要发生在第 2 和第 3 外显子的 12、13 和 61 密码子。
从 58 例口腔癌和 106 例结直肠癌患者的 DNA 样本中分离出来。采用两对用于第 2 和第 3 外显子的通用引物，在单个管中对三个 RAS 基因的第 2 外显子和第 3 外显子的 12 和 13 密码子以及第 61 密码子进行多重扩增。将产物在三个管中进行清洗和分离。分别使用 7 种不同大小的 RAS 引物针对不同的 RAS 基因，对每个 RAS 基因的 12、13 和 61 密码子进行引物延伸，以检测碱基变化。
我们将这两种方法在 58 例口腔癌和 106 例结直肠癌中检测 N-、H-和 KRAS 突变的结果进行了比较。两种方法的结果一致，但我们的方法在工作量和所需时间方面优于直接测序。
我们提出了一种快速检测人类癌症中 N-、H-和 KRAS 基因的 12、13 和 61 密码子突变的方法。
