Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry.

L A Haff; I P Smirnov

doi:10.1101/gr.7.4.378

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Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry.

L A Haff·I P Smirnov

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Abstract

We report a simple method, the PinPoint assay, for detecting and identifying single-base variations (polymorphisms) at specific locations within DNA sequences. An oligonucleotide primer is annealed to the target DNA immediately upstream of the polymorphic site and is extended by a single base in the presence of all four dideoxynucleotide triphosphates and a thermostable DNA polymerase. The extension products are desalted, concentrated, and subjected to delayed-extraction MALDI-TOF mass spectrometry. The base at the polymorphic site is identified by the mass added onto the primer. Heterozygous targets produce two mass-resolved species that represent the addition of both bases complementary to those at the polymorphic site. The assay is suitable for double-stranded PCR products without purification or strand separation. More than one primer can be simultaneously extended and then mass-analyzed. The mass spectrometric method thus shows promise for high-volume diagnostic or genotyping applications.

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BRCA1 Protein Base Sequence DNA DNA Primers DNA-Directed DNA Polymerase Deoxyribonucleotides Enzyme Stability Genetic Techniques Heterozygote Molecular Sequence Data Nucleotides Polymerase Chain Reaction Polymorphism, Genetic Sensitivity and Specificity Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Taq Polymerase beta-Galactosidase Comparative Study Journal Article

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L A Haff, I P Smirnov. 1997. Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry.. https://doi.org/10.1101/gr.7.4.378

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