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Nucleic Acids Research, 2000, Vol. 28, No. 12 E68-e68
© 2000 Oxford University Press

A new MALDI-TOF based mini-sequencing assay for genotyping of SNPS

Xiyuan Sun1,2, H. Ding1, K. Hung1 and Baochuan Guo1,*

1Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA and 2Institute of Genetics, Fudan University, Shanghai 200433, China

A new MALDI-TOF based mini-sequencing assay termed VSET was developed for genotyping of SNPs. In this assay, specific fragments of genomic DNA containing the SNP site(s) are first amplified, followed by mini-sequencing in the presence of three ddNTPs and the fourth nucleotide in the deoxy form. In this way, the primer is extended by only one base from one allele, while it is typically extended by two bases from another allele. The products are then analyzed using MALDI-TOF mass spectrometry. The genotype of the SNP site is identified based on the number of nucleotides added. This assay has been examined using both synthetic and genomic DNA samples. In addition, multiplexed assays were successfully performed to genotype four SNP sites in a single tube. The main aspect of this assay is that it can overcome the key problems associated with the currently used mini-sequencing methods. First, it significantly reduces the stringent high-resolution and extensive desalting requirements that are essential to the pinpoint assay. Second, it avoids the long extension problem associated with the PROBE assay.

* To whom correspondence should be addressed. Tel: +1 216 687 2471; Fax: +1 216 687 9298; Email: b.guo@csuohio.edu


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