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Published online 24 February 2004

Nucleic Acids Research, 2004, Vol. 32, No. 4 e42
© 2004 Oxford University Press

Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system

Binzhi Ren, Jing-Min Zhou and Makoto Komiyama*

Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan

*To whom correspondence should be addressed. Tel: +81 3 5452 5200; Fax: +81 3 5452 5209; Email: komiyama{at}mkomi.rcast.u-tokyo.ac.jp
+AF261279

Single-nucleotide polymorphisms (SNPs) in double-stranded DNA (dsDNA) have been straightforwardly genotyped by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). Peptide nucleic acid (PNA), a DNA analog, was used as a probe molecule. In its presence, genomic dsDNA was first treated with exonuclease III and then with nuclease S1. By these one-pot reactions, single-stranded DNA fragments including the SNP sites were formed in situ. These fragments were directly analyzed by MALDI-TOF MS, and the identity of the DNA base at the SNP site was determined in terms of mass number. By using two or more PNA probes simultaneously, multiplex analysis was also successful. Various genotypes of apolipoprotein E gene ({epsilon}2/{epsilon}2, {epsilon}3/{epsilon}3, {epsilon}4/{epsilon}4, {epsilon}2/{epsilon}3 and {epsilon}3/{epsilon}4) were identified from dsDNA obtained by PCR from corresponding patients.


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