Brief Article
Copyright ©2010 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Oct 14, 2010; 16(38): 4858-4864
Published online Oct 14, 2010. doi: 10.3748/wjg.v16.i38.4858
Comparative analysis of dideoxy sequencing, the KRAS StripAssay and pyrosequencing for detection of KRAS mutation
Jing Gao, Yan-Yan Li, Ping-Nai Sun, Lin Shen
Jing Gao, Yan-Yan Li, Ping-Nai Sun, Lin Shen, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of GI Oncology, Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing 100142, China
Author contributions: Gao J, Li YY and Sun PN performed the experiments; Gao J wrote the manuscript; Shen L designed the experiments and revised the manuscript.
Correspondence to: Lin Shen, Professor, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of GI Oncology, Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing 100142, China. lin100@medmail.com.cn
Telephone: +86-10-88196561 Fax: +86-10-88196561
Received: April 19, 2010
Revised: June 24, 2010
Accepted: July 1, 2010
Published online: October 14, 2010
Abstract

AIM: To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients.

METHODS: Formalin-fixed, paraffin-embedded (FFPE) samples with tumor cells ≥ 50% were collected from 100 Chinese CRC patients at Beijing Cancer Hospital. After the extraction of genome DNA from FFPE samples, fragments contained codons 12 and 13 of KRAS exon 2 were amplified by polymerase chain reaction and analyzed by dideoxy sequencing, the KRAS StripAssay and pyrosequencing. In addition, the sensitivities of the 3 methods were compared on serial dilutions (contents of mutant DNA: 100%, 50%, 20%, 15%, 10%, 5%, 1%, 0%) of A549 cell line DNA (carrying the codon 12 Gly>Ser mutation) into wild-type DNA (human normal intestinal mucosa). The results of dideoxy sequencing, the KRAS StripAssay and pyrosequencing were analyzed by Chromas Software, Collector for KRAS StripAssay and the pyrosequencing PyroMarkTM Q24 system, respectively.

RESULTS: Among 100 patients, KRAS mutations were identified in 34%, 37% and 37% of patients by dideoxy sequencing, the KRAS StripAssay and pyrosequencing, respectively. The sensitivity was highest with the KRAS StripAssay (1%), followed by pyrosequencing (5%), and dideoxy sequencing was lowest (15%). Six different mutation types were found in this study with 3 main mutations Gly12Asp (GGT>GAT), Gly12Val (GGT>GTT) and Gly13Asp (GGC>GAC). Thirty-three patients were identified to have KRAS mutations by the 3 methods, and a total of 8 patients had conflicting results between 3 methods: 4 mutations not detected by dideoxy sequencing and the KRAS StripAssay were identified by pyrosequencing; 3 mutations not detected by dideoxy sequencing and pyrosequencing were identified by the KRAS StripAssay; and 1 mutation not detected by pyrosequencing was confirmed by dideoxy sequencing and the KRAS StripAssay. Among these discordant results, the results identified by dideoxy sequencing were consistent either with the KRAS StripAssay or with pyrosequencing, which indicated that the accuracy of dideoxy sequencing was high.

CONCLUSION: Taking a worldwide view of reports and our results, dideoxy sequencing remains the most popular method because of its low cost and high accuracy.

Keywords: DNA mutational analysis; KRAS; Mutation; Dideoxy sequencing; KRAS StripAssay; Pyrosequencing