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 description/ionization mass spectrometric (MALDI MS) detection using a nitrocellulose film substrate. This method employs polymerase chain reaction (PCR) amplification using DNA extracted from buccal cells as templates, followed by direct digestion with restriction enzymes and subsequent analysis by MALDI MS. The extraction of DNA from buccal cells provides a rapid and convenient means for sampling PCR-based diagnostic analysis. The amount of DNA was sufficient as the template for both normal PCR amplifications, and amplifications involving the use of mismatched primers and multiple primers. The MALDI MS methodology has been successfully used for the analysis of such PCR products where restriction fragments generated directly in PCR reactions have been used for detection of carbonic anhydrase and cystic fibrosis transmembrane conductance regulator as model genes. The detection of genetic polymorphisms following routine biological and clinical procedures with the MALDI MS method is demonstrated. The results from MALDI MS analysis are shown to be comparable to those obtained from gel electrophoresis but the MALDI MS method is several orders of magnitude faster than gel electrophoretic techniques. The method described herein should also be readily extended to other areas involving DNA screening and testing.
Liu
Y H
YH
Department of Chemistry, University of Michigan, Ann Arbor 48109, USA.
Bai
J
J
Zhu
Y
Y
Liang
X
X
Siemieniak
D
D
Venta
P J
PJ
Lubman
D M
DM
D001483:Base Sequence; D003101:Collodion; D004247:DNA / Q000737:chemistry*; D004262:DNA Restriction Enzymes; D006801:Humans; D007834:Lasers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009061:Mouth Mucosa / Q000737:chemistry* / Q000166:cytology; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic
D003101:Collodion; D004247:DNA; D004262:DNA Restriction Enzymes