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Nucleic Acids Research, 2000, Vol. 28, No. 5 1193-1198
© 2000 Oxford University Press

DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides

Andrey A. Stomakhin1, Vadim A. Vasiliskov1,2, Edward Timofeev1,3,4, Dennis Schulga1,2, Richard J. Cotter4 and Andrei D. Mirzabekov1,3,2,*

1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 117984 Moscow, Russia, 2Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Russia, 3Biochip Technology Center, Argonne National Laboratory, Argonne, IL 60439, USA and 4Middle Atlantic Mass Spectrometry Laboratory, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA–8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10–15°C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA–8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contigu­ously stacked 5mers to increase the length of DNA for sequence analysis is discussed.

* To whom correspondence should be addressed at: Biochip Technology Center, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439-4833, USA. Tel: +1 630 252 3161; Fax: +1 630 252 9155; Email: amir@everest.bim.anl.gov


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