Highly sensitive nonradioactive single-strand conformational polymorphism. Detection of Ki-ras mutations.
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.
Emanuel
J R
JR
Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023, USA.
Damico
C
C
Ahn
S
S
Bautista
D
D
Costa
J
J
D004247:DNA / Q000032:analysis*; D004252:DNA Mutational Analysis / Q000379:methods*; D004586:Electrophoresis; D005456:Fluorescent Dyes; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C098798:SYBR Green II; D004247:DNA