CHARACTERIZATION OF PCR PRODUCTS FROM BACILLI USING ELECTROSPRAY-IONIZATION FTICR MASS-SPECTROMETRY

A procedure for rapid purification of polymerase chain reaction (PCR) products allowing precise molecular weight determination using electro spray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is described, PCR amplification utilized the DNA po lymerase from Pyrococcus furiosus (Pfu) which, unlike Tag, does not in corporate a nontemplated terminal deoxyadenosine phosphate, An 89-base pair nucleotide portion of the spacer region between the 16S and 23S ribosomal rRNA genes was amplified from the genome of three members of Bacillus cereus group and a 114 nucleotide region from the Bacillus s ubtilis, PCR involves polymerization of nucleotide precursors using tw o oligonucleotide primers and an amplification enzyme, as well as the presence of metal ions, Mass spectrometric analysis greatly benefits f rom removal of the oligonucleotide primers (15- and 17-mers in this in stance) and nucleotide precursors since they adversely affect sensitiv ity and metal ion adduction results in an inaccurate molecular weight determination, In the presence of guanidinium hydrochloride, the PCR p roducts bind preferentially to a silica resin, allowing removal of oth er components (i,e,, dNTP's, primers, and salts), Further removal of m etal ions was accomplished using a microdialysis device, allowing samp les to be pumped through a hollow cellulose fiber with an external cou ntercurrent now of 2.5 mM ammonium acetate, Prior to injection into th e mass spectrometer, the sample buffer was adjusted to 50 vol % aceton itrile, 25 mM piperidine, and 25 mM imidazole, which enhanced signal i ntensity, The molecular weights of the PCR products determined by nucl eotide sequence and MS analysis were in excellent agreement, and sever al PCR products were analyzed where mass differences corresponding to single base substitutions could be accurately assigned, These assignme nts were possible due to the high mass precision, accuracy, and resolu tion FTICR inherently affords, This constitutes the first report demon strating the ionization and detection of PCR products by mass spectrom etry with mass precision and accuracy for assigment of such modificati ons or substitutions.