Real-time allele-specific amplification for sensitive detection of the BRAF mutation V600E

被引:81
作者
Jarry, A
Masson, D
Cassagnau, E
Parois, S
Laboisse, C
Denis, MG
机构
[1] Inst Biol, Lab Biochim Specialisee, F-44093 Nantes, France
[2] INSERM, U539, Fac Med, F-44035 Nantes, France
[3] Inst Biol, Lab Anat Pathol, F-44093 Nantes, France
关键词
BRAF; mutation; allele specific PCR; real time PCR;
D O I
10.1016/j.mcp.2004.05.004
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
BRAF is a cytoplasmic serine/threonine kinase in the MAPK pathway that transduces signals from RAS family members to MEK1/2. Mutations in the BRAF gene have been described in the majority of cutaneous melanomas, papillary thyroid carcinoma and to a lesser extent in other cancers. The predominant mutation reported is a single transversion in exon 15 (T1799A). We designed a real-time allele-specific PCR to detect this mutation. This assay allowed us to detect this alteration in samples containing 2% of cells harboring this mutation, which is equivalent to 1 % mutated DNA, assuming heterozygosity for the allele. Using this assay, we then tested 44 human primary colorectal tumors. We found the V600E mutation in four samples (9.1 %). Analysis of DNA extracted from paraffin-embedded sections gave similar results, indicating that archived tissues can also be analyzed using this assay. The advantages of this method include: (1) rapidity; (2) sensitivity; (3) ease; (4) large throughput; (5) low cost and (6) the small quantities of DNA needed. (C) 2004 Elsevier Ltd. All rights reserved.
引用
收藏
页码:349 / 352
页数:4
相关论文
共 11 条
  • [1] Lack of BRAF mutation in primary uveal melanoma
    Cohen, Y
    Goldenberg-Cohen, N
    Parrella, P
    Chowers, I
    Merbs, SL
    Pe'er, J
    Sidransky, D
    [J]. INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 2003, 44 (07) : 2876 - 2878
  • [2] Mutations of the BRAF gene in human cancer
    Davies, H
    Bignell, GR
    Cox, C
    Stephens, P
    Edkins, S
    Clegg, S
    Teague, J
    Woffendin, H
    Garnett, MJ
    Bottomley, W
    Davis, N
    Dicks, N
    Ewing, R
    Floyd, Y
    Gray, K
    Hall, S
    Hawes, R
    Hughes, J
    Kosmidou, V
    Menzies, A
    Mould, C
    Parker, A
    Stevens, C
    Watt, S
    Hooper, S
    Wilson, R
    Jayatilake, H
    Gusterson, BA
    Cooper, C
    Shipley, J
    Hargrave, D
    Pritchard-Jones, K
    Maitland, N
    Chenevix-Trench, G
    Riggins, GJ
    Bigner, DD
    Palmieri, G
    Cossu, A
    Flanagan, A
    Nicholson, A
    Ho, JWC
    Leung, SY
    Yuen, ST
    Weber, BL
    Siegler, HF
    Darrow, TL
    Paterson, H
    Marais, R
    Marshall, CJ
    Wooster, R
    [J]. NATURE, 2002, 417 (6892) : 949 - 954
  • [3] Gorden A, 2003, CANCER RES, V63, P3955
  • [4] Kimura ET, 2003, CANCER RES, V63, P1454
  • [5] Kumar R, 2003, CLIN CANCER RES, V9, P3362
  • [6] Raf proteins and cancer: B-Raf is identified as a mutational target
    Mercer, KE
    Pritchard, CA
    [J]. BIOCHIMICA ET BIOPHYSICA ACTA-REVIEWS ON CANCER, 2003, 1653 (01): : 25 - 40
  • [7] BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas
    Nikiforova, MN
    Kimura, ET
    Gandhi, M
    Biddinger, PW
    Knauf, JA
    Basolo, F
    Zhu, ZW
    Giannini, R
    Salvatore, G
    Fusco, A
    Santoro, M
    Fagin, JA
    Nikiforov, YE
    [J]. JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, 2003, 88 (11) : 5399 - 5404
  • [8] Tumorigenesis -: RAF/RAS oncogenes and mismatch-repair status
    Rajagopalan, H
    Bardelli, A
    Lengauer, C
    Kinzler, KW
    Vogelstein, B
    Velculescu, VE
    [J]. NATURE, 2002, 418 (6901) : 934 - 934
  • [9] Rimoldi D, 2003, CANCER RES, V63, P5712
  • [10] Wang L, 2003, CANCER RES, V63, P5209