Nucleic acid diagnostics based on mass spectrometry or mass separation and base specific cleavage

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated in part on a base specific cleavage reaction to generate a set of small oligonucleotides bounded by the base cleaved.
The nucleic acid molecule may be completely or only partially cleaved or digested.
These fragments are then separated based on mass by MALDI-TOF MS.
This generates a fingerprint of the nucleic acid fragment comprising a series of peaks where each peak represents the mass of each small cleavage product.
As a result of the sensitivity of mass determination, each oligonucleotide of given length but different nucleotide composition produces a different mass.
The mass of each peak, therefore, corresponds to the nucleotide composition of the fragment as well as to its length.
Consequently, any nucleotide substitution results in either a shifted peak due to the mass difference in the new cleavage fragment or, if the mutation changes the targeted base, a cleavage product containing a different number of bases.
Accordingly, one aspect of the present invention contemplates a method of detecting a difference of one or more nucleotides between a nucleic acid molecule to be tested and a reference nucleic acid molecule, said method comprising subjecting the test nucleic acid molecule to base specific cleavage to generate oligonucleotide fragments, separating the resulting oligonucleotide fragments based on mass by MALDI-TOF MS and/or other equivalent procedure to produce a fingerprint of the oligonucleotide fragments comprising one or more peaks wherein a peak represents the mass of each fragment and identifying an altered peak relative to a reference nucleic acid molecule subjected to the same procedure wherein the presence of an altered peak is indicative of a difference of one or more nucleotides in said tested nucleic acid molecule.
Conveniently, screening is carried out by comparing the cleavage product masses of the reference or wild-type nucleic acid to those of the test sample.
Mass changes corresponding to base changes are readily observed