DNA-SEQUENCE ANALYSIS BY MALDI MASS-SPECTROMETRY

Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products, Gel casting and electrophoresis are the t ime limiting steps, and the gel separation is occasionally imperfect d ue to aberrant mobility of certain fragments, leading to erroneous seq uence determination, Furthermore, illegitimately terminated products f requently cannot be distinguished from correctly terminated ones, a ph enomenon that also obscures data interpretation. Ire the present work the base of MALDI mass spectrometry for sequencing of DNA amplified fr om clinical samples is implemented, The unambiguous and fast identific ation of deletions and substitutions in DNA amplified from heterozygou s carriers realistically suggest MALDI mass spectrometry as a future a lternative to conventional sequencing procedures for high throughput s creening for mutations. Unique features of the method are demonstrated tay sequencing a DNA fragment that could not be sequenced conventiona lly because of gel electrophoretic band compression and the presence o f multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the seq uence was deduced, and the nature of the non-specific termination coul d be determined. The method described here increases the fidelity ire DNA sequencing, is fast, compatible with standard DNA sequencing proce dures, and amenable to automation.