High-throughput SNP genotyping by combining exonuclease III, nuclease S1, and acridine-bearing PNA.
Recently we reported a precise and straightforward method for genotyping of single nucleotide polymorphisms (SNPs) in double-stranded DNA substrates. The substrates were consecutively treated by two enzymes (exonuclease III and nuclease S1) in the presence of peptide nucleic acid (PNA) probes, and the resultant DNA fragments were analyzed by mass spectroscopy. Here, the selectivity of DNA scission by nuclease S1 has been greatly improved by attaching an acridine to the PNA probes. Modification at the C-terminus was especially effective. The number of DNA fragments formed has been greatly decreased so that double-stranded DNA substrates can be genotyped by using the two enzymes still more easily and precisely.
Ren
Binzhi
B
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
Komiyama
Makoto
M
D000166:Acridines / Q000378:metabolism*; D001057:Apolipoproteins E / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000166:Acridines; D001057:Apolipoproteins E; D004277:DNA, Single-Stranded; D020135:Peptide Nucleic Acids; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D015719:Single-Strand Specific DNA and RNA Endonucleases