DOE Human Genome Program Contractor-Grantee
39. Stable Isotope Assisted Mass Spectrometry Allows Accurate Determination of Nucleotide Compositions of PCR Products
Xian Chen1, Zhengdong Fei2, Lloyd M. Smith2, E. Morton Bradbury3,4, and Vahid Majidi1
1CST-9, Chemical Science and Technology Division and 3B-3, MS M888, Biological Division, Los Alamos National Laboratory, Los Alamos, NM 87544; 2Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706-1396; and 4Department of Biological Chemistry, School of Medicine, University of California at Davis, Davis, CA 95616
In parallel with the large-scale sequencing effort, the human genome project will need the next generation tools for accurate and efficient analyses of the enormous pool of DNA sequences. Such analyses 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. Here, we report a novel method that combines stable isotope 13C/15N-labeling of PCR products of the target sequences with analysis of the mass shifts by mass spectrometry (MS). The mass-shift due to the labeling of a single type of nucleotide (i.e., A, T, G, or C) will reveal the number of that type of nucleotide in a given DNA fragment. Using this technique, we have accurately determined nucleotide compositions of DNA fragments. The method has also been applied to score a known single nucleotide polymorphism. The comparisons of nucleotide compositions determined by our method among homologous sequences are useful in sequence validation, sequence comparison, and characterizations of sequence polymorphisms.
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