Detection of base pair mismatches in duplex DNA and RNA oligonucleotides using electrospray mass spectrometry

Richard H. Griffey; Michael J. Greig

doi:10.1117/12.274340

22 May 1997 Detection of base pair mismatches in duplex DNA and RNA oligonucleotides using electrospray mass spectrometry

Richard H. Griffey, Michael J. Greig

Author Affiliations +

Proceedings Volume 2985, Ultrasensitive Biochemical Diagnostics II; (1997) https://doi.org/10.1117/12.274340
Event: BiOS '97, Part of Photonics West, 1997, San Jose, CA, United States

Abstract

The identify and location of base pair mismatches in non- covalent DNA:RNA duplexes are established using MS and MS-MS on a quadruple ion trap with electrospray ionization (ESI). MS-MS experiments on a 14mer duplex (D) with a single C:A base pair mismatch using lower activation energy results in selective cleavage of the mismatched A nucleobase, even in the presence of the wild-type duplex. The location of the mismatch base pair can be discerned via presence of the wild-type duplex. The location of the mismatch base pair can be discerned via selection of the (D-5H)^5- ion and fragmentation of the backbone at that location in a n additional MS-MS experiment. Selective fragmentation is observed for C in a C-C mismatched base pair, which is very difficult to detect using chemical cleavage or E. coli mismatch binding protein. In an RNA:DNA duplex with a single base pair mismatch, the DNA base is removed without fragmentation of the RNA strand, greatly simplifying the interpretation of the resulting MS spectrum. A method is presented for detecting two DNA strands, for example a point mutation which generates an oncogenic phenotype, and the wild-type message. The results suggest that ESI-MS-MS may provide a rapid and selective method to identify and locate genetic mutations without the need for chemical degradation or protein binding followed by gel electrophoresis.

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Richard H. Griffey and Michael J. Greig "Detection of base pair mismatches in duplex DNA and RNA oligonucleotides using electrospray mass spectrometry", Proc. SPIE 2985, Ultrasensitive Biochemical Diagnostics II, (22 May 1997); https://doi.org/10.1117/12.274340

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