Chris E. Hopkins, Mark Leppert, Jim McCloskey, Pamela Crain, and Raymond Gesteland
Department of Human Genetics, University of Utah, Salt Lake City, UT, 84112
Electrospray Mass Spectrometry (EMS) enables very accurate molecular weight determinations, to less than 0.01% error. In recent years EMS has enabled the discovery of novel and rare modifications of ribonucleic acids, and has enabled mass validation of tRNAs up to 120 bases long. EMS analysis is rapid, typically within ten minutes per assay, and reliable, due accurate mass determination.
The EMS technique has great potential for detecting single base differences in genomic PCR amplification products. A primer pair is designed to flank a polymorphic locus. The primers, separated by one to four bases, will generate upon amplification one product from each chromosome. In a heterozygote the two PCR products will differ by at least the identity of one base pair. The mass analysis of the denatured mixture will give the molecular weight of the four strands and thus define the identity of the polymorphism.
To demonstrate this capability, PCR EMS has been applied to identifying a known C to T polymorphism in the alpha 4 subunit of the nicotinic acytylcholine receptor gene (CH alpha 4), a candidate gene for several forms of human epilepsy. A 53 base pair region encompassing this polymorphism was amplified from a pair of primers, 24 and 25 bases in length. The 4 bases between the primers contain the C to T polymorphic site. Mass analysis of this site on a heterozygotic individual generates mass data on each of the strands amplified from the paired chromosomes. The accuracy is within 0.01% mass error compared to theoretical molecular weight, thus allowing unambigous identification of the polymorphism.
Synthetic templates have been generated to model all possible point polymorphisms at this locus. Mass data generated from these models demonstrates that all are readily detectable. This technique will enable sequence variant determination to made in hours instead of days and it should be applicable to any PCR amplified product up to a theoretical and practical limit of 70 base pairs for point polymorphisms and up to 150 for base deletion polymorphisms. It is expected that this technique will be a valuable tool for DNA diagnostic analysis.
*Supported by a grant from the Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-FG94ER61817