Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry.
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.
Haff
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PerSeptive Biosystems, Framingham, Massachusetts 01701, USA. Ihaff@pbio.com
Smirnov
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D019313:BRCA1 Protein / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D005821:Genetic Techniques; D006579:Heterozygote; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D019914:Taq Polymerase; D001616:beta-Galactosidase / Q000235:genetics
D019313:BRCA1 Protein; D017931:DNA Primers; D003854:Deoxyribonucleotides; D009711:Nucleotides; D004247:DNA; C072146:Pfu DNA polymerase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D001616:beta-Galactosidase