Patent ID: 6303298
Filing Date: 2001-10-16
Classification: C12Q,Y10T

Abstract:
A method for PCR amplification of selected DNA segments and preparation of these samples for mass spectrometric analysis using matrix assisted laser desorption/ionization (MALDI), whereby the DNA is modified such that the DNA segments become fragmentation-stable, easy to ionize and inert against adduct formation in the MALDI process, comprising the following steps:(1) amplifying DNA segments using unmodified primers and unmodified substrates by normal PCR to produce sufficient DNA segments for a subsequent MALDI analysis,(2) exposing the amplified DNA segments to a modified primer having a predetermined charge suitable for analysis using MALDI, and at least a first substrate suitable for forming electrically neutral linkages, wherein the modified primer bonds only to one strand of any given DNA segment,(3) performing at least one enzymatic replication with the modified primer and at least the first substrate to generate modified segments of DNA analogs, the replication proceeding such that modified DNA strands form double strands with unmodified DNA strands,(4) preparing MALDI sample spots containing the modified DNA segments on a sample support, and(5) analyzing the molecular weight of the DNA segments by MALDI mass spectrometry.