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 procedures 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 substrate. In this method, on-probe purification allows for effective elimination of the interfering effects of salts, buffers, and other contaminants that are usually present in DNA samples, which serve as important limiting factors in the DNA molecular ion yield in the MALDI process. The use of the nitrocellulose film substrate also appears to improve the shot-to-shot and sample-to-sample reproducibility of the ion yield due to the more homogeneous coverage of matrix/analyte over the sample surface. With the use of the nitrocellulose substrate, DNA fragments of up to 622 base pairs in complex mixtures provide mass spectra with minimal sample purification. Although only species corresponding to single-stranded DNA were detected, a mass calibration scheme was established allowing an accuracy of within one base pair for fragments of < 250 bp under the experimental conditions. Despite the low mass resolution of the spectra obtained, the method has been successfully used for rapid DNA screening for sample disease genes and PCR products.
Liu
Y H
YH
Department of Chemistry, University of Michigan, Ann Arbor 48109-1055, USA.
Bai
J
J
Liang
X
X
Lubman
D M
DM
Venta
P J
PJ
D001483:Base Sequence; D003101:Collodion; D004247:DNA / Q000032:analysis*; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping / Q000295:instrumentation*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003101:Collodion; D004247:DNA