Colorimetric approach to high-throughput mutation analysis.
High-throughput genomic mutation screening for primary tumors has characteristically been expensive, labor-intensive, and inadequate to detect low levels of mutation in a background of wild-type signal. We present a new, combined PCR and colorimetric approach that is inexpensive, simple, and can detect the presence of 1% mutation in a background of wild-type. We compared manual dideoxy sequencing of p53 for eight lung cancer samples to a novel assay combining a primer extension step and an enzymatic colorimetric step in a 96-well plate with covalently attached oligonucleotide sequences. For every sample, we were able to detect the presence or absence of the specific mutation with a statistically significant difference between the sample optical density (OD) and the background OD, with a sensitivity and specificity of 100%. This assay is straightforward, accurate, inexpensive, and allows for rapid, high-throughput analysis of samples, making it ideal for genomic mutation or polymorphism screening studies in both clinical and research settings.
Benoit
Nicole E
NE
Johns Hopkins University School of Medicine, Baltimore MD 21205, USA.
Goldenberg
David
D
Deng
Shirley X
SX
Rosenbaum
Eli
E
Cohen
Yoram
Y
Califano
Joseph A
JA
Shackelford
William H
WH
Wang
Xiao B
XB
Sidransky
David
D
D001681:Biological Assay; D003124:Colorimetry / Q000379:methods*; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D009154:Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D017931:DNA Primers; D004273:DNA, Neoplasm; D016159:Tumor Suppressor Protein p53