Development of New High Performance Liquid Chromatographic and Mass Spectrometric Techniques for Allelic Discrimination

Erwin Schrödinger Fellowship J 1922 Development of new techniques for allelic discrimination Andreas PREMSTALLER 08.05.2000 The detection and identification of DNA sequence variations is one of the key steps in genomic analysis. Especially point mutations represent important diagnostic markers. The ability to efficiently discover and genotype single nucleotide polymorphisms is increasingly seen as fundamental to future molecular genetic analysis of inherited diseases, and in particular, of common complex diseases caused by more than one mutation. At present, there are many techniques available for comparative DNA sequencing, but with rapidly increasing DNA sequence information available for humans and other species, there is a clear need for methods that have high throughput, are sensitive, and are at least semi-automated. Denaturing high-performance liquid chromatography (DHPLQ can efficiently reveal single mismatches in amplified PCR samples ranging in size from 80-2000 base pairs. Highly efficient chromatographic separation systems in combination with partial heat denaturation of the DNA fragments enable the easy resolution of homo- and heteroduplexes within minutes. With the exception of very AT- or GC- rich sequences, a single set of conditions is sufficient to discover all point mutations as well as deletions and insertions independent of their location. Mass spectrometry as detection technique will broaden the range of applications of DHPLC for allelic discrimination. The primary goal of this project is the elaboration of new techniques for allelic discrimination employing denaturing liquid chromatographic separation methods coupled to mass spectrometry. Application of electrospray ionization mass spectrometry as detection technique for ion-pair reversed-phase high-performance liquid chromatography under completely denaturing conditions will allow the accurate and fast detection and genotyping of mutations. Furthermore, mass infon-nation will facilitate the typing of mutations that can not be discriminated by denaturing high-performance liquid chromatography with traditional optical detectors. The inherently low throughput of chromatographic analysis will be significantly enhanced by multiplexed analysis of a multitude of samples using photocleavable mass spectrometry tags to distinguish between samples related to different individuals or different loci. Construction of a dedicated system incorporating the chromatographic separation of multiple PCR products, the on-line photocleavage of the mass spectrometry tags, followed by on-line mass analysis using atmospheric pressure chemical ionization mass spectrometry will result in a dramatic increase in the sample throughput for routine mutation screening. The developed techniques will contribute to an improved understanding of human disease and evolution and specifically find applications in the field of clinical diagnosis of breast cancer genes which help to assess the hereditary risk of breast cancer in women.