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Albitar M, Wu WI, Feltz E, et al. Simplified Reverse Dot Blot Analyses for Detecting of ras Oncogene Mutations. Mol Diagn. 1997;2(3):169-176doi: 10.1054/MODI00200169.
Albitar, M., Wu, W. I., Feltz, E., Jin, G., Hirsch-Ginsberg, C., Kantarjian, H., & Beran, M. (1997). Simplified Reverse Dot Blot Analyses for Detecting of ras Oncogene Mutations. Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology, 2(3), 169-176. https://doi.org/10.1054/MODI00200169
Albitar, et al. "Simplified Reverse Dot Blot Analyses for Detecting of ras Oncogene Mutations." Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology vol. 2,3 (1997): 169-176. doi: https://doi.org/10.1054/MODI00200169
Albitar M, Wu WI, Feltz E, Jin G, Hirsch-Ginsberg C, Kantarjian H, Beran M. Simplified Reverse Dot Blot Analyses for Detecting of ras Oncogene Mutations. Mol Diagn. 1997 Sep;2(3):169-176. doi: 10.1054/MODI00200169. PMID: 10462606.
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Mol Diagn. 1997 Sep;2(3):169-176. doi: 10.1054/MODI00200169.

Simplified Reverse Dot Blot Analyses for Detecting of ras Oncogene Mutations.

Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology

Albitar, Wu, Feltz, Jin, Hirsch-Ginsberg, Kantarjian, Beran

Affiliations

  1. Section of Hematopathology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA

PMID: 10462606 DOI: 10.1054/MODI00200169

Abstract

Background: Mutations in members of the ras gene family (H-ras, K-ras, and N-ras) have been identified in various human malignancies. A variety of techniques have been used to test for ras mutations. Methods and Results: A simplified reverse dot blot (RDB) assay was used in this study. Polymerase chain reaction products were hybridized to nitrocellulose membrane-fixed synthetic probes (20 nucleotides long) specific for codons 12, 13, and 61 of H-, K-, and N-ras mutations and their wild-type sequences. No special treatment or modification of the probes was necessary to obtain adequate results in overnight film exposure when the polymerase chain reaction was carried out using (32)P-end labeled primers. It was demonstrated that this simplified RDB assay can also be used with fluorescein-11-dUTP and a chemiluminescence detection system. The RDB assay is more reliable than the single-strand conformation polymorphism (SSCP) assay. By comparison, the SSCP assay is significantly less sensitive and less specific. It was confirmed with sequencing that 11 (12%) of 93 SSCP assays were false positive and 2 (2%) were false negative, whereas no false positive or false negative RDB assay was detected. The RDB assay also provides more additional detailed information about the specific point mutation and amino acid change, which may have clinical implications in some tumors. Conclusions: The RDB assay is very sensitive and able to detect mutations when the mutant allele is in 1% of the cells and can be used to detect minimal residual disease, particularly in some cases of leukemia and myelodysplasia.

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