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

Genome Res. 1997 Apr;7(4):378-88. doi: 10.1101/gr.7.4.378.


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.

Publication types

  • Comparative Study

MeSH terms

  • BRCA1 Protein / genetics
  • Base Sequence
  • DNA / chemistry
  • DNA / genetics
  • DNA Primers / chemistry
  • DNA Primers / genetics
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • Deoxyribonucleotides / chemistry
  • Deoxyribonucleotides / genetics
  • Enzyme Stability
  • Genetic Techniques*
  • Heterozygote
  • Molecular Sequence Data
  • Nucleotides / chemistry
  • Nucleotides / genetics
  • Polymerase Chain Reaction
  • Polymorphism, Genetic*
  • Sensitivity and Specificity
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
  • Taq Polymerase
  • beta-Galactosidase / genetics


  • BRCA1 Protein
  • DNA Primers
  • Deoxyribonucleotides
  • Nucleotides
  • DNA
  • Pfu DNA polymerase
  • Taq Polymerase
  • DNA-Directed DNA Polymerase
  • beta-Galactosidase