Throughout this application, various publications are referenced in parentheses. Citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Single nucleotide polymorphisms (SNPs), the most common genetic variations in the human genome, are important markers for identifying disease genes and for pharmacogenetic studies (1, 2). SNPs appear in the human genome with an average density of once every 1000-base pairs (3). To perform large-scale SNP genotyping, a rapid, precise and cost-effective method is required. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (4) allows rapid and accurate sample measurements (5–7) and has been used in a variety of SNP detection methods including hybridization (8–10), invasive cleavage (11, 12) and single base extension (SBE) (5, 13–17). SBE is widely used for multiplex SNP analysis. In this method, primers designed to anneal immediately adjacent to a polymorphic site are extended by a single dideoxynucleotide that is complementary to the nucleotide at the variable site. By measuring the mass of the resulting extension product, a particular SNP can be identified. Current SBE methods to perform multiplex SNP analysis using MS require unambiguous simultaneous detection of a library of primers and their extension products. However, limitations in resolution and sensitivity of MALDI-TOF MS for longer DNA molecules make it difficult to simultaneously measure DNA fragments over a large mass range (6). The requirement to measure both primers and their extension products in this range limits the scope of multiplexing.
A high fidelity DNA sequencing method has been developed which uses solid phase capturable biotinylated dideoxynucleotides (biotin-ddNTPs) by detection with fluorescence (18) or mass spectrometry (19), eliminating false terminations and excess primers. Combinatorial fluorescence energy transfer tags and biotin-ddNTPs have also been used to detect SNPs (20).
False stops or terminations occur when a deoxynucleotide rather than a dideoxynucleotide terminates a sequencing fragment. It has been shown that false stops and primers which have dimerized can produce peaks in the mass spectra that can mask the actual results preventing accurate base identification (21).
The present application discloses an approach using solid phase capturable biotin-ddNTPs in SBE for multiplex genotyping by MALDI-TOF MS. In this method primers that have different molecular weights and that are specific to the polymorphic sites in the DNA template are extended with biotin-ddNTPs by DNA polymerase to generate 3′-biotinylated DNA extension products. The 3′-biotinylated DNAs are then captured by streptavidin-coated magnetic beads, while the unextended primers and other components in the reaction are washed away. The pure DNA extension products are subsequently released from the magnetic beads, for example by denaturing the biotin-streptavidin interaction with formamide, and analyzed with MALDI-TOF MS. The nucleotide at the polymorphic site is identified by analyzing the mass difference between the primer extension product and an internal mass standard added to the purified DNA products. Since the primer extension products are isolated prior to MS analysis, the resulting mass spectrum is free of non-extended primer peaks and their associated dimers, which increases the accuracy and scope of multiplexing in SNP analysis. The solid phase purification system also facilitates desalting of the captured oligonucleotides. Desalting is critical in sample preparation for MALDI-TOF MS measurement since alkaline and alkaline earth salts can form adducts with DNA fragments that interfere with accurate peak detection (21).