USE OF A NITROCELLULOSE FILM SUBSTRATE IN MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY FOR DNA MAPPING AND SCREENING/

A nitrocellulose film is used as a substrate in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) studies of DNA. PCR products and DNA fragment digests obtained from biochemical procedure s can be analyzed with the use of a nitrocellulose substrate in MALDI MS whereas no signal is observed with the use of a stainless steel sub strate. In this method, on-probe purification allows for effective eli mination of the interfering effects of salts, buffers, and other conta minants that are usually present in DNA samples, which serve as import ant limiting factors in the DNA molecular ion yield in the MALDI proce ss. The use of the nitrocellulose film substrate also appears to impro ve the shot-to-shot and sample-to-sample reproducibility of the ion yi eld due to the more homogeneous coverage of matrix/analyte over the sa mple surface. With the use of the nitrocellulose substrate, DNA fragme nts of up to 622 base pairs in complex mixtures provide mass spectra w ith minimal sample purification. Although only species corresponding t o single-stranded DNA were detected, a mass calibration scheme was est ablished allowing an accuracy of within one base pair for fragments of <250 bp under the experimental conditions. Despite the low mass resol ution of the spectra obtained, the method has been successfully used f or rapid DNA screening for sample disease genes and PCR products.