Mass Spectrometry for Forensic Applications

Recent advances in DNA technology for the detection of variation in specific DNA sequences have proved that DNA fingerprinting analysis can become a very valuable technology for forensic applications. By analyzing a sufficient number of regions of DNA that show variability between nonrelated individuals, one can reduce the probability of two individuals matching by chance, to practically zero. Thus, it can be demonstrated that an individual's genetic fingerprint is as unique as their skin fingerprints, and often easier to obtain. However, the requirements for DNA fingerprinting for forensic application involve not only the technology but also the cost, time and reliability of the analysis. DNA fingerprinting technology has tremendous power of exclusion and inclusion and is used routinely as legal evidence. Due to the high reliability of DNA fingerprinting, courts often require DNA analysis as evidence in the most serious criminal cases. However, present gel electrophoresis analysis of DNA in forensic applications is considered to be slow and expensive. Consequently, a backlog of DNA analysis for forensics is common. Mass spectrometry DNA analysis can help to resolve this difficulty, since it can be faster, cheaper and more reliable for forensic sample analysis. DNA fingerprinting is a technology that identifies individuals based on patterns of DNA markers detected in the genomic DNA. It can be used in forensic analysis to identify suspects or victims. It is particularly valuable at scenes of violent crimes where a body may not be available, or in instances where decomposition or dismemberment excludes the use of standard forensic techniques. There are several different approaches available to obtain DNA fingerprints for forensic application. These include restriction fragment length polymorphism (RFLP), short tandem repeats (STRs), single-nucleotide polymorphism (SNP) and DNA sequencing. Currently, the bulk of RFLP, STR, SNP analysis and DNA sequencing utilize gel electrophoresis, which is time-consuming and labor intensive. With the newly developed mass spectrometry technology for DNA analysis, the analysis time can possibly be reduced by more than 90% and automation of sample analysis is much easier. Furthermore, mass spectrometric analysis does not require the use of chemical dyes or radioactive materials, thus increasing safety and reducing cost in sample preparation and handling. In addition, the cost and concern of disposing of hazardous waste are eliminated. The major difficulties preventing the use of mass spectrometry for DNA analysis are the very low vapor pressure of DNA and the fragmentation of these large DNA molecules by most ionization methods. In 1987, Professors Hillenkamp, Karas and their colleagues successfully developed matrix-assisted laser desorption/ionization (MALDI) for protein analysis. In this approach, a protein sample was mixed with a much larger quantity of small organic compounds which serve as a matrix. These matrixes, such as sinapinic acid, functioned to prevent the protein molecules from becoming entangled with one another. In MALDI, the DNA sample is irradiated with a laser beam, which causes desorption. The wavelength of the laser is often chosen so as to have strong absorption by the small matrix molecules, but not the protein. After the absorption of the laser photons by the matrix molecules, the matrix molecules are vaporized and carry the much larger protein molecules into space. Some ions and electrons are also produced due to this laser ablation process. Some protein ions can be produced by protonation or deprotonation processes caused by the interaction of desorbed protein molecules and desorbed matrix molecules or ions. During the past few years, MALDI has also been successfully applied to DNA detection through the discovery of new matrixes and improvements in instrumentation. The main advantage of MALDI analysis is its very fast speed. However, the size of DNA fragments which can be detected is still limited to less than 3000 base pairs (bp). Mass spectra for DNA fragments larger than 100 nucleotides usually have poor mass resolution (M/ΔM < 200). Nevertheless, there are many forensic applications using mass spectrometry for DNA analysis which are not limited by modest mass resolution and detection efficiency of large DNA fragments.