DNA Analysis by Coupled Separation-Mass Spectrometry

DNA analysis has its roots in classical genetics, biochemistry, molecular biology, and medicinal diagnostic. However, it has recently found itself in an unlikely area: a court of law. In Austria, every few week newspapers report on crime cases that could be solved on the basis of DNA analysis. The rapidly growing importance of DNA analysis for forensic science is not only the result of integration of DNA data into large DNA databases but also a result of the fast development of new analytical technologies to generate DNA data. Liquid phase separation techniques such as chromatography and electrophoresis have always played key role in the separation and detection of nucleic acids. During the past decade, mass spectrometry has become another important tool in the analysis of nucleic acids mainly because of the introduction of new soft ionization methods such as electrospray ionization and matrix-assisted laser desorption ionization, which allow the intact ionization and detection of large biomolecules well exceeding 500 kD. However, the mass spectrometric analysis of nucleic acids is associated with difficulties arising from their polyanionic nature and tendency to form quite stable adducts with alkali cations resulting in mass spectra of poor quality. The purity of the nucleic acid sample to be introduced into a mass spectrometer is another critical factor for successful mass spectrometric analysis. Consequently, purification of nucleic acids is very important to obtain mass spectra of high quality and information content. Miniaturization and on-line conjugation of the purification methods to mass spectrometry is obligatory to be able to handle the minute amounts of nucleic acids available for forensic DNA analysis. The primary goal of this project is to develop new miniaturized liquid phase separation methods to be used as on- line sample concentration, desalting, purification, and separation techniques that can be readily coupled to electrospray and matrix-assisted laser desorption mass spectrometry in order to allow sensitive detection and characterization of nucleic acid samples. The proposed approach to analyze nucleic acids consists of a combination of different technologies which are 1. synthesis of polymeric stationary phase materials of continuous-bed morphology; 2. utilization of continuous-bed capillary columns for miniaturized liquid phase separation techniques such as capillary liquid chromatography and capillary electrochromatography; and 3. on-line conjugation of separation methods to electrospray and matrix-assisted laser desorption/ionization mass spectrometry. The miniaturization of the separation systems is accomplished through the synthesis and application of capillary columns with a stationary phase of continuous-bed morphology with inner diameters of 50--360 micro-m. Resulting eluent flow rates in the range of 0.1-10 micro-L/min are optimally suited for on-line coupling with electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry. Two different miniaturized liquid phase separation methods that offer different levels of separation efficiency and selectivity but also entail different levels of complexity and sophistication are optimized for coupling to mass spectrometry. The separation of complex nucleic acid mixtures such as DNA restriction digests and multiplex polymerase chain reaction products is achieved by capillary liquid chromatography and capillary electrochromatography. The chromatographic mode proposed for separation of nucleic acids is ion-pair reversed-phase chromatography with a hydrophobic, polymeric stationary phase and a mobile phase containing water, acetonitrile and volatile ion-pair reagents (triethylammonium bicarbonate, triethylammoniurn hexafluoroisopropanolate). The developed and optimized analytical techniques will be applied to the analysis of oligonucleotides, DNA fragments and PCR products related to polymorphic DNA of relevance for forensic science. National and international cooperations are available and will be used to ensure efficient application and evaluation of the new analytical methods.

Accurate molecular mass measurements of nucleic acids enable the generation of valuable data on genomic sequences in biological samples. However, this method was so far only of limited applicability to real samples, e. g. of forensic relevance, since such samples contain only very small amounts of material. Financially supported by the project P-14133-PHY of the Austrian Science Fund, the team around the analytical chemist Christian Huber of the Institute of Analytical Chemistry and Radiochemistry, Innsbruck, in collaboration with Walther Parson from the Institute of Legal Medicine, Innsbruck, and Peter Oefner from the Stanford Genome Technology Center, California, now succeeded in analyzing minimal amounts of DNA by using liquid chromatography mass spectrometry. Improvements mainly in sample preparation now make mass spectrometry amenable to micro traces containing as little as 100 picograms of DNA. The newly developed method represents a major step forward towards alternative methods for forensic and genetic DNA characterization. Although the past few years have seen a number of new technologies, most of them were not successful because they were not sensitive or specific enough to be able to analyze real samples. In forensic analysis, for instance, usually the length of DNA molecules is determined using capillary electrophoresis, in order to prove a connection between traces of DNA found at a scene of crime and a suspect. Instead of the length of DNA, mass spectrometry investigates rapidly and with high accuracy the molecular mass of DNA molecules. High purity of the sample is a prime prerequisite for successful molecular mass measurements. For this purpose, liquid chromatography has been optimized as purification method, in which the sample is pumped through a porous polymer material. DNA is retained on the polymer, whereas impurities pass. Subsequently, the DNA is transferred directly from the porous material into a mass spectrometer for molecular mass determination. Although the differences between the genomes of two individuals are very subtle (one in approx. 500-20000 Bases), they are sufficient for the differentiation of individuals on the basis of so-called DNA profiles. On a statistical basis, at least one billion individuals have to be investigated to accidentally find two identical DNA profiles. With the help of DNA profiles, it is possible to convict suspects with high confidence and to unequivocally exclude innocent persons. The major advantages of liquid chromatography-mass spectrometry technology rely in the high precision of the data, their high information content, the high degree of automation of the whole process, and the high sample throughput, which is indispensable in connection with the rapidly expanding DNA databases