Highly sensitive nonradioactive single-strand conformational polymorphism. Detection of Ki-ras mutations - PubMed (original) (raw)
Highly sensitive nonradioactive single-strand conformational polymorphism. Detection of Ki-ras mutations
J R Emanuel et al. Diagn Mol Pathol. 1996 Dec.
Abstract
Mutation detection by single-strand conformational polymorphism (SSCP) analysis is more difficult when the variant is limited to a small proportion of target sequences in a sample. Use of SYBR-Green II, a sensitive, nonradioactive, minimally hazardous nucleic acid stain, permits detection of Ki-ras mutants present as less than 0.5% of the target sequences. The polymerase chain reaction (PCR) primers we have selected produce an amplicon that distinguishes all clinically observed variants in Ki-ras codons 12 and 13 from the wild type. We compared mutant discrimination and SYBR-Green II detection sensitivity in three formats: (a) standard MDE gel SSCP, (b) rapid minigel MDE using an internal gel temperature controller, and (c) rapid resolution in chilled 15% (37.5:1) acrylamide minigels. All these gels are easily evaluated by standard ultraviolet transillumination and digital image analysis. This ssDNA staining method is rapid, highly reproducible, and minimally hazardous, and minigels use 25% the reagents of most other systems. Our improvements are relevant for the detection of mutations in pathologic samples with minimal targets, such as fine-needle aspirates, and body fluids in which mutated alleles of a gene may be present at low levels but carry a high level of diagnostic or prognostic importance.
Similar articles
- A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue.
Sarkar FH, Valdivieso M, Borders J, Yao KL, Raval MM, Madan SK, Sreepathi P, Shimoyama R, Steiger Z, Visscher DW, et al. Sarkar FH, et al. Diagn Mol Pathol. 1995 Dec;4(4):266-73. doi: 10.1097/00019606-199512000-00007. Diagn Mol Pathol. 1995. PMID: 8634783 - The utility of Ki-ras mutation analysis in the cytologic diagnosis of pancreatobiliary neoplasma.
Dillon DA, Johnson CC, Topazian MD, Tallini G, Rimm DL, Costa JC. Dillon DA, et al. Cancer J. 2000 Sep-Oct;6(5):294-301. Cancer J. 2000. PMID: 11079168 - K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis.
Korn SH, Moerkerk PT, de Goeij AF. Korn SH, et al. J Clin Pathol. 1993 Jul;46(7):621-3. doi: 10.1136/jcp.46.7.621. J Clin Pathol. 1993. PMID: 8157747 Free PMC article. - PCR-based approaches for detection of mutated ras genes.
van Mansfeld AD, Bos JL. van Mansfeld AD, et al. PCR Methods Appl. 1992 May;1(4):211-6. doi: 10.1101/gr.1.4.211. PCR Methods Appl. 1992. PMID: 1362126 Review. No abstract available.
Cited by
- Sensitive sequencing method for KRAS mutation detection by Pyrosequencing.
Ogino S, Kawasaki T, Brahmandam M, Yan L, Cantor M, Namgyal C, Mino-Kenudson M, Lauwers GY, Loda M, Fuchs CS. Ogino S, et al. J Mol Diagn. 2005 Aug;7(3):413-21. doi: 10.1016/S1525-1578(10)60571-5. J Mol Diagn. 2005. PMID: 16049314 Free PMC article. - Detection of clonal T-cell receptor gamma gene rearrangements in paraffin-embedded tissue by polymerase chain reaction and nonradioactive single-strand conformational polymorphism analysis.
Signoretti S, Murphy M, Cangi MG, Puddu P, Kadin ME, Loda M. Signoretti S, et al. Am J Pathol. 1999 Jan;154(1):67-75. doi: 10.1016/s0002-9440(10)65252-2. Am J Pathol. 1999. PMID: 9916920 Free PMC article. - High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer.
Krypuy M, Newnham GM, Thomas DM, Conron M, Dobrovic A. Krypuy M, et al. BMC Cancer. 2006 Dec 21;6:295. doi: 10.1186/1471-2407-6-295. BMC Cancer. 2006. PMID: 17184525 Free PMC article. - KRAS genotyping of paraffin-embedded colorectal cancer tissue in routine diagnostics: comparison of methods and impact of histology.
Weichert W, Schewe C, Lehmann A, Sers C, Denkert C, Budczies J, Stenzinger A, Joos H, Landt O, Heiser V, Röcken C, Dietel M. Weichert W, et al. J Mol Diagn. 2010 Jan;12(1):35-42. doi: 10.2353/jmoldx.2010.090079. Epub 2009 Dec 10. J Mol Diagn. 2010. PMID: 20007841 Free PMC article. - Absence of Ras, c-myc and Epidermal Growth Factor Receptor (EGFR) Mutations in Human Gliomas and its Clinical Factors Associated with Pathological Grading After Six Years of Diagnosis in North East Malaysian Patients.
Ghazali MM, Mohd Zan MS, Yusof AA, Abdullah JM, Jaffar H, Ariff AR, Mar Slamah W, Ideris A, Ali AM, Omar AR, Yuosff K, Mohd Lila MA, Othman F, Mustapha NM, Isa MN, Naing NN. Ghazali MM, et al. Malays J Med Sci. 2005 Jul;12(2):27-33. Malays J Med Sci. 2005. PMID: 22605955 Free PMC article.
MeSH terms
Substances
LinkOut - more resources
Full Text Sources