Importance

Loading Importance Indicators...

Abstract

The determination of base (nucleotide) composition in DNA by mass spectrometry is described. Accurate and efficient analyses of the enormous pool of DNA sequences are required for; (a) validation of DNA sequences; (b) comparison of a parent (known) sequence with a related (unknown) sequence, and (c) characterization of sequence polymorphisms in various genes especially those associated with genetically inherited human diseases. The combination of stable isotope-labeling of PCR products of target sequences with analysis of the mass shifts by mass spectrometry (MS) is shown to provide such analyses, since the mass-shift due to the labeling of a single type of nucleotide (i.e., A, T, G, or C) identifies the number of that type of nucleotide in a given DNA fragment. Accurate determinations of nucleotide compositions of DNA fragments have been achieved with an accuracy of .+-.0.03% with respect to their known sequences. The method has also been applied to identify a known single-nucleotide polymorphism (SNP). The comparisons of nucleotide compositions determined according to the teachings of the present method among homologous sequences are useful in sequence validation, sequence comparison, and characterizations of sequences polymorphisms.

Loading the Abstract Image...

First Claim