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Proceedings Paper

Detection of base pair mismatches in duplex DNA and RNA oligonucleotides using electrospray mass spectrometry
Author(s): Richard H. Griffey; Michael J. Greig
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Paper 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.

Paper Details

Date Published: 22 May 1997
PDF: 5 pages
Proc. SPIE 2985, Ultrasensitive Biochemical Diagnostics II, (22 May 1997); doi: 10.1117/12.274340
Show Author Affiliations
Richard H. Griffey, Isis Pharmaceuticals (United States)
Michael J. Greig, Isis Pharmaceuticals (United States)

Published in SPIE Proceedings Vol. 2985:
Ultrasensitive Biochemical Diagnostics II
Gerald E. Cohn; Gerald E. Cohn; Steven A. Soper, Editor(s)

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