Our work was the first to demonstrate that base substitutions, deletions or additions between two polymerase chain reaction (PCR) products can be determined (within 30 sec) by accurate measurements of molecular weight (MW) using mass spectrometry (MS). Alternatively, tandem mass spectrometry (MS/MS) provides much greater discriminating power since a """"""""fingerprint"""""""" of oligonucleotide fragments of the PCR products are generated.
Aim 1. is designed to demonstrate that MS/MS fingerprints (using low resolution ion trap or triple quadrupole instruments) correlate with sequencing using electrophoresis and that fingerprints will fall into a limited number of patterns, reflecting genetic polymorphism. For initial application of our PCR-MS/MS approach to the detection of variability in human genetic material, we chose the p53 gene. We will begin by using PCR-MS/MS for the detection of p53 mutations in Burkitt's - lymphoma and colon cancer cell lines and primary cultures of normal human fibroblasts.
Aim 2 involves parallel studies with both low resolution and high resolution instruments to extend the power of MS/MS finger-printing. As with restriction digests and SSCP, there are likely to be situations where certain PCR products with different sequences may produce fingerprints indistinguishable by simple visual observation. The strength of MS/MS (unlike other fingerprinting techniques) is that nucleotide base composition (not sequence) can be readily obtained for each oligonucleotide fragment of the parent PCR molecule. Using FTICR, the charge and mass/charge will be determined for each fragment in MS/MS fingerprints of PCR products. This will allow the MW to be calculated for each fragment. It will then be possible to determine the number of A,C,G and Ts in each fragment (using a low resolution instrument) by analyzing PCR products labelled with one of each of 4 stable isotope labelled nucleotides and observing the mass change of rebelled fragments versus the unlabelled. Pairs of PCR products generated from the rRNA interspace region of known sequence (from wild type strains and those modified by site directed mutagenesis) of B.subtilis and other bacilli differing by single and multiple base changes will be analyzed. Finally the results of Aim 1 and 2 will be integrated to perform a final MS/MS fingerprinting study (including determining the mass and A,C,G and T content of oligonucleotide fragments).
