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A novel single-base extension (SBE) assay using cleavable and noncleavable SBE primers in the same reaction mix is described. The cleavable SBE primers consisted of deoxyribonucleotides and one ribonucleotide (hereafter denoted chimeric primers), whereas the noncleavable SBE primers consisted of only deoxyribonucleotides (hereafter denoted standard primers). Biotin-labeled ddNTPs were used in the SBE reaction, and the SBE products were purified using the monomeric avidin triethylamine purification protocol, ensuring that only primers extended with a biotin-ddNTP in the 3'-end were isolated. A ribonuclease mix was developed to specifically cleave the chimeric primers, irrespective of the base of the ribonucleotide, whereas standard primers without a ribonucleotide were unaffected by the ribonuclease treatment. The SBE products were analyzed in linear mode using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The cleaved SBE products were detected in the 2000-5500 m/z range, and the noncleaved SBE products were detected in the 5500-10 000 m/z range. The method was validated by typing 17 Y chromosome single-nucleotide polymorphisms in 100 males with a 17-plex SBE package containing 9 chimeric primers and 8 standard primers.

One of the most promising techniques for typing of multiple single-nucleotide polymorphism (SNP) is detection of single base extension primers (SBE) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a new MALDI-TOF MS protocol for typing of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle conditions using 5% triethylamine. We dubbed this method monomeric avidin triethylamine purification. The biotin-labeled ddNTPs contained linkers with different masses ensuring a clear separation of the alleles even for SBE primers with a mass of 10 300 Da. Furthermore, only 25-350 fmol of SBE primers were necessary in order to obtain reproducible MALDI-TOF spectra. Similar signal intensities were obtained in the 5500-10 300 m/z mass range by increasing the concentration of the longer SBE primers in the reaction. To validate the technique, 17 Y-chromosome SNPs were analyzed in 200 males. The precision and accuracy of the mass determination were analyzed by parametric statistic, and the potential use of MALDI-TOF MS for SNP typing is discussed.

Double-stranded DNA ranging from 9 kDa to over 500 kDa were desorbed and analyzed by MALDI TOF mass spectrometry. IR-MALDI with glycerol as matrix yielded excellent results for larger double-stranded DNA by adjustment of the ionic strength through the addition of salts. Very little fragmentation and a routine sensitivity in the subpicomole range were observed in IR-MALDI when double-stranded analytes harboring 70 base pairs or more were probed. In the lower mass range (up to approximately 70 base pairs), UV-MALDI with 6-aza-2-thiothymine as matrix was the ionization method of choice because it allowed specific double-stranded complexes containing relatively few base pairs to be desorbed intactly. In this mode, an essentially quantitative detection of the double-stranded form was observed for a 70-mer. The UV-MALDI was accompanied by a significant fragmentation and a resulting reduced sensitivity and mass resolution. The methods described open MALDI-MS for the analysis of large DNA-DNA and DNA-protein complexes.