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Nucleic Acids Research, 2003, Vol. 31, No. 9 e47
© 2003 Oxford University Press

RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis

Ralf Hartmer, Niels Storm, Sebastian Boecker1, Charles P. Rodi2, Franz Hillenkamp3, Christian Jurinke1 and Dirk van den Boom1

SEQUENOM GmbH, Mendelssohnstrasse 15D, D-22761 Hamburg, Germany, 1 SEQUENOM Inc., 3595 John Hopkins Court, San Diego, CA 92121, USA, 2 Rodi Pharma, Inc., 13823 Recuerdo Drive, Del Mar, CA 92014, USA and 3 Institute for Medical Physics and Biophysics, University of Münster, Robert-Koch-St 31, D-48149 Münster, Germany

*To whom correspondence should be addressed. Tel: +1 858 202 9066; Fax: +1 858 202 9084; Email: dvandenboom{at}sequenom.com

Here we devise a new method for high-throughput comparative sequence analysis. The developed protocol comprises a homogeneous in vitro transcription/RNase cleavage system with the accuracy and data acquisition speed of matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS). In summary, the target region is PCR amplified using primers tagged with promoter sequences of T7 or SP6 RNA polymerase. Using RNase T1, the in vitro transcripts are base-specifically cleaved at every G-position. This reaction results in a characteristic pattern of fragment masses that is indicative of the original target sequence. To enable high-throughput analysis, samples are processed with automated liquid handling devices and nanoliter amounts are dispensed onto SpectroCHIP arrays for reliable and homogeneous MALDI preparation. This system enables rapid automated comparative sequence analysis for PCR products up to 1 kb in length. We demonstrate the feasibility of the devised method for analysis of single nucleotide polymorphisms (SNPs) and pathogen identification.


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