RAPID SCREENING OF GENETIC POLYMORPHISMS USING BUCCAL CELL-DNA WITH DETECTION BY MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY

A new approach is developed for the rapid and cost-effective detection of human genetic polymorphisms based on matrix-assisted laser descrip tion/ionization mass spectrometric (MALDI MS) detection using a nitroc ellulose film substrate. This method employs polymerase chain reaction (PCR) amplification using DNA extracted from buccal cells as template s, followed by direct digestion with restriction enzymes and subsequen t analysis by MALDI MS. The extraction of DNA from buccal cells provid es a rapid and convenient means for sampling PCR-based diagnostic anal ysis. The amount of DNA was sufficient as the template for both normal PCR amplifications, and amplifications involving the use of mismatche d primers and multiple primers. The MALDI MS methodology has been succ essfully used for the analysis of such PCR products where restriction fragments generated directly in PCR reactions have been used for detec tion of carbonic anhydrase and cystic fibrosis transmembrane conductan ce regulator as model genes. The detection of genetic polymorphisms fo llowing routine biological and clinical procedures with the MALDI MS m ethod is demonstrated. The results from MALDI MS analysis are shown to be comparable to those obtained from gel electrophoresis but the MALD I MS method is several orders of magnitude faster than gel electrophor etic techniques. The method described herein should also be readily ex tended to other areas involving DNA screening and testing.