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Nucleic Acids Research, 2001, Vol. 29, No. 3 e11
© 2001 Oxford University Press

DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry

Young-Soo Kwon, Kai Tang1, Charles R. Cantor1, Hubert Köster1 and Changwon Kang*

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea and 1Sequenom, Inc., 11555 Sorrento Valley Road, San Diego, CA 92121, USA

Sets of RNA ladders can be synthesized by transcription of a bacteriophage-encoded RNA polymerase using 3'-deoxynucleotides as chain terminators. These ladders can be used for sequencing of DNA. Using a nicked form of phage SP6 RNA polymerase in this study substantially enhanced yields of transcriptional sequencing ladders. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of chain-terminated RNA ladders allowed DNA sequence determination of up to 56 nt. It is also demonstrated that A->G and C->T variations in heterozygous and homozygous samples can be unambiguously identified by the mass spectrometric analysis. As a step towards single-tube sequencing reactions, {alpha}-thiotriphosphate nucleotide analogs were used to overcome problems caused by chain terminator-independent, premature termination and by the small mass difference between natural pyrimidine nucleotides.

* To whom correspondence should be addressed. Tel: +82 42 869 2628; Fax: +82 42 869 2682; Email: ckang{at}mail.kaist.ac.kr


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