As the DNA sequence of the human genome is now completed and available, interest is shifting towards the detection of DNA in candidate genes of human diseases. These DNA modifications can be either in the sequence or epigenetic. Thus, rare, frequent, known and unknown polymorphisms can be identified through development of new methods of sequencing for each individual. We have developed a new method for DNA analysis using cleavage of RNA/DNA chimera and MALDI-TOF MS. The method takes advantage of a novel class of thermostable DNA polymerases that also incorporate ribonucleotides. The RNA/DNA chimera contains three deoxyribonucleotides and the fourth nucleotide in its ribonucleotide form. These products can be either single-stranded linear or double-stranded. The RNA/DNA chimera is cleaved by sodium hydroxide after each ribonucleotide base. All fragments contain only one ribonucleotide 3' terminal base with a phosphate terminal group. Fragments are desalted by the addition of cation exchange resin and masses are determined by MALDI-TOF MS. The mass fingerprint is used to identify deviations from the reference sequence. Gas phase fragmentations of the RNA/DNA chimera were also studied. With the high throughput capability of MALDI-TOF MS, this facile method is well suited for screening DNA sequences of limited genomic regions of interest in a large number of individuals. This rapid and accurate, new method, has many potential applications including: DNA genotyping, DNA multiplex allele-specific genotyping, DNA micro-haplotyping, DNA methylation analysis and DNA sequencing
