Lead Inventor:
Jingyue Ju, Ph.D.
Single Nucleotide Polymorphisms Important Gene Markers:
Single nucleotide polymorphisms (SNPs) are the most common genetic variations in the human genome, occurring approximately every thousand base pairs. These base mismatches serve as important markers for gene identification, so numerous methods have been developed for large-scale SNP genotyping. Among these methods, single base extension (SBE), coupled with matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF), is particularly useful for rapid and precise SNP analysis. Although MALDI-TOF can detect a library of DNA primers and their extension products, this technique suffers from 1) limited resolution and sensitivity for longer DNA molecules and 2) false signals from extension byproducts. Therefore, increasing the accessible mass range of MALDI-TOF and eliminating false signals would greatly improve the multiplexing scope of SBE/MALDI-TOF genotyping.
Large-Scale Genotyping Free of Byproduct Signals:
This invention describes an improved approach to MALDI-TOF/SBE based genotyping using solid phase-capturable, biotin-modified didedoxy nucleotide triphosphates (biotin-ddNTPs). In this method, each of the four possible biotin ddNTPs (A, G, C, or T) is designed to have a different molecular weight. Primers specific to a polymorphic site in a DNA template are extended by a biotin-ddNTP that is complementary to the nucleotide at the variable site. This generates a 3'-biotinylated DNA extension product that has a unique molecular weight and is then captured via streptavidin-coated magnetic beads. Subsequently, the reaction byproducts are washed away, the captured extension products are chemically/photolytically released from the beads, and the nucleotide at the desired site is identified via internally standardized MALDI-TOF. This method not only yields a mass spectrum that is free of byproduct signals but can also accurately analyze longer DNA strands due to careful calibration with an internal mass standard.
Applications:
• General genotyping between members of the same species
• Human genetic fingerprinting for identity testing
• Rapid identification of genes that underlie cancer and other diseases
• Improved drug discovery for pharmacogenomics and chemogenomics
Advantages:
• More accurate identification of SNPs in longer DNA sequences
• Elimination of false positives from template extension byproducts
• Compatibility with solid supports and microarrays for massively parallel SNP genotyping
• High degree of modularity and flexibility through variation of the biotin-ddNTP structures
Patent Status: Patent (
US 7074597 B2)
Licensing Status: Available for Licensing and Sponsored Research Support
Publications:
Kim S, Edwards JR, Deng L, Chung W, and Ju J. Solid Phase Capturable Dideoxynucleotides for Multiplex Genotyping Using Mass Spectrometry (2002) Nucl. Acids Res. 30: e85