Single-Nucleotide Polymorphism Identification Assays Using a Thermostable DNA Polymerase and Delayed Extraction MALDI-TOF Mass Spectrometry | Zendy

Lawrence A. Haff | Zendy; Igor P. Smirnov | Zendy

Open Access

Single-Nucleotide Polymorphism Identification Assays Using a Thermostable DNA Polymerase and Delayed Extraction MALDI-TOF Mass Spectrometry

Author(s) -

Lawrence A. Haff,

Igor P. Smirnov

Publication year - 1997

Publication title -

genome research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.556

H-Index - 297

eISSN - 1549-5469

pISSN - 1088-9051

DOI - 10.1101/gr.7.4.378

Subject(s) - biology , primer (cosmetics) , genotyping , oligonucleotide , primer extension , microbiology and biotechnology , mass spectrometry , polymerase chain reaction , dna , dna extraction , nucleotide , chromatography , primer dimer , genetics , multiplex polymerase chain reaction , genotype , chemistry , base sequence , gene , organic chemistry

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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