PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection.
By the combination of peptide nucleic acid (PNA) with single-stranded DNA specific nucleases, alteration of a single base to another in DNA has been detected with high accuracy. Only the DNAs in DNA/PNA duplexes involving a mismatch are efficiently hydrolyzed by these enzymes, whereas fully matching sequences are kept intact. This difference is visually scored by adding 3,3'-diethylthiadicarbocyanine, which changes its color from blue to purple upon binding to DNA/PNA duplexes. These findings are applied to the convenient and straightforward detection of single nucleotide polymorphisms (SNPs). When the target site in the sample DNA is completely complementary with the PNA, a notable amount of DNA/PNA duplex remains and thus the solution exhibits purple color. In the presence of even one mismatch between PNA and DNA, however, the DNA is completely digested by the enzyme and therefore the dye shows its intrinsic blue color. The SNPs in the apolipoprotein E gene of human DNA have been successfully genotyped by this method.
Komiyama
Makoto
M
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Japan. komiyama@mkomi.rcast.u-tokyo.ac.jp
Ye
Sheng
S
Liang
Xingguo
X
Yamamoto
Yoji
Y
Tomita
Takafumi
T
Zhou
Jing-Min
JM
Aburatani
Hiroyuki
H
D001057:Apolipoproteins E / Q000235:genetics*; D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D020135:Peptide Nucleic Acids; D020641:Polymorphism, Single Nucleotide; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
D001057:Apolipoproteins E; D020135:Peptide Nucleic Acids; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases