Highly sensitive nonradioactive single-strand conformational polymorphism. Detection of Ki-ras mutations

Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B
J R EmanuelJ Costa

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 ...Continue Reading

Citations

Jul 29, 2005·The Journal of Molecular Diagnostics : JMD·Shuji OginoCharles S Fuchs
Jun 22, 2001·Experimental and Molecular Pathology·B R DoolittleJ Costa
Nov 3, 2004·European Journal of Cancer : Official Journal for European Organization for Research and Treatment of Cancer (EORTC) [and] European Association for Cancer Research (EACR)·Tanja PejovicKunle Odunsi
Aug 30, 2003·Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology·Ginesa Garcia-RostanGiovanni Tallini
Sep 4, 1999·Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B·M E CouceD Carter
Jun 5, 2002·Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B·Giovanna Maria CrisiGiovanni Tallini
Dec 10, 2014·Expert Review of Molecular Diagnostics·Sudhir Perincheri, Pei Hui

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