Analysis of DNA using MALDI-TOF mass spectrometry.

摘要

Novel modifications were developed for the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for DNA analysis in various clinical/diagnostic areas. Although there are many, genotyping single point mutations, and detection of cancer cells are two types of appropriate applications of these modifications. A hydrophobic membrane of Parafilm and Teflon was substituted for the conventional stainless steel probe tip as a novel sample preparation method to improve the DNA analysis using MALDI-TOF. This method can solve two major problems encountered in the MALDI-TOF, namely poor detection limits and strong salt effects. Advantages of this method includes: (1) increased detection limits (5 to 10 times for a 85mer), and improved detection of larger DNA components in a DNA mixture; (2) increased salt tolerance limits, especially for larger DNA; (3) achievement of an excellent mass resolution similar to that observed using a metal probe for DNA up to 62mer; (4) effective analysis of salt-contaminated protein samples; and (5) elimination of steps required for the cleaning of probes after analysis.; A point mutation G1691 → A in the coagulation Factor V gene, results in a Arg506 → Gln amino acid mutation in the Factor V molecule. This mutation defined as Factor VLEIDEN, leads to an activated protein C (APC)-resistance and is the most common genetic risk factor for familial thrombophilia. A MALDI-TOF based mini-sequencing method, a fragment of genornic DNA containing the 1691th base is first amplified, followed by mini-sequencing conducted in the presence of dGTP, and ddATP, ddCTP, and ddTTP. The extended products are then analyzed using MALDI-TOF mass spectrometry. The base at the position 1691 is identified based on the number of nucleotides added. This method can genotype 16 APC-resistance patients previously identified by conventional methods and 11 normal control samples in a blinded manner. The genotypes of all samples were correctly identified. This method is accurate, fast, and allows for simultaneous multiplex genotyping of a number of mutation sites.