Simultaneous detection of multiple single nucleotide polymorphism by single-strand-specific nuclease and PNA probe

02003 Oxford University Press ~~ Nucleic Acids Research Supplement No. 3 185-186 ~ Simultaneous detection of multiple single nucleotide polymorphism by single-strand-specific nuclease and PNA probe Sheng Ye, Xingguo Liang, Yoji Yamamoto, Jing-Min Zhou, Takafumi Tomita, Hiroyuki Aburatani and Makoto Komiyama Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan protected from the nuclease when the DNA was complementary to the PNA. With even one mismatch between them, however, the DNA was almost completely digested. This difference could be visualized with Disc@). Thus, alteration of a single nucleotide in DNA can be detected even with our naked eyes. INTRODUCTION Scheme 1. Schematic illustration of the method €or visual SNP detection. Single nucleotide polymorphisms are the most frequent type of variation in the human genome, and are powerful tools for a variety of medical and genetic studies. Thus precise and economical methods for S N P detection are crucially important and necessary. Although a number of methods have been reported,’’ no one can meet all the requirements (cheapness, promptness, preciseness and others). Recently, we reported an effective and facile method for S N P detection by combining single-strand specific nuclease (nuclease S1, Mung Bean nuclease, and nuclease P1) and PNA.3’4As schematically depicted in Scheme 1, the target DNA sequence was almost completely DNA samples - - full-match I ,/ *,, Nuclease treatment ..‘* DiSc,(S) mismatch I mononucleotide -.,,. 1 ‘4 Purple Blue RESULTS AND DISCUSSION The SNPs within codon 112 (TGC-CGC) and codon 158 (GGC-TGC) of the human apoE gene are risk factors for the development of Alzheimer’s disease. Four 80-mer single-stranded DNA encompassing either the apoE codon 112 or 158 (named Codon 112 TGC/CGC or 158 CGCnGC) were chemically synthesized. The target sequences are either perfectly complementary to the PNA probe in Table 1, or contain a single nucleotide mismatch upon the formation of P N m N A duplexes. ABSTRACT The combination of PNA (peptide nucleic acid) and single-strand-specific nuclease have been used for detection of single nucleotide polymorphisms (SNPs). When DNA is perfectly complementary to PNA, it is protected from digestion by the nuclease. If there exists a single-base mismatch between them, however, the DNA is completely digested. These differences are visualized by using 3,3’-diethylthiadicarbocyanine (DiSc2(5)), which changes its color from blue to purple upon binding to PNA/DNA hybrids. In terms of this methodology, homozygous and heterozygous SNPs in apoE gene have been successfully analyzed. Furthermore, the multiplex SNPs are simultaneously genotyped This technique provides a simple, straightforward, hcile, and visual genetic screening, with no need for expensive and complicated equipment. Nucleic Acids Research Supplement No. 3 186 0.6 Table 1. The sequences of DNA samples and PNA probes. PNA probes DNA Codon 112 TGC PNA1: TGCACACGCC Codon 112 CGC PNA2: TGCACKGCC Codon 158 CGC PNA3: TClTCGCGGA Codon158TGC PNA4: TCnCACGGA ~~~~ ~~ ~ Abs 0.2 0 -0.1 400 ' I I I I 500 600 700 800 Wavelenqthlnml Figure 1. Detection of upoE SNP codon 112 by using PNAl probe. f/f, homozygousCodon 112 TGC (without mismatch); m/m, homozygous Codon 112 CGC (with a mismatch): f/m, heterozygous DNA (the 1:l mixture of the above two DNAs). (DNAld.0 pM, [PNA1]=1.2 yM, [nuclease Sl] =1.S UIpL at 25 OC for 20 min, pH 4.6; the enzymatic digests were stained with 20yM DiSc,(S). Table 2. Simultaneous analysis of two SNPs by using four PNA probes. PNAl PNA2 P N M PNA4 Codon 112 TGC Purple Blue Codon 112 CGC Blue Purple Codon 158 CGC Purple Blue Codon 158 TGC Blue Pumle ACKNOWLEDGEMENTS The authors thank Prof. Hisakazu Mihara (the Tokyo Institute of Technology) for his kind assistance in the preparation of PNA. This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. REFERENCES Syvanen, A.-C. (2001) Nature Rev. Genet. 2, 930-942. Gut, I.<;. (2001) Hum. Mutat. 17, 475-492. Ye, S.; Liang, X.G.; Yamamoto, Y.; Komiyama, M. (2002) Nucleic Acids Res. Supple. 2,235-236. Komiyama, M.; Ye, S.; Liang, XG.; Yamamoto, Y.; Tomita, T.; Zhou, J.-M.; Aburatani, H. (2003)l. Am. Chem. SOC.125, 3758-3762. The target sequence in Codon 112 TGC is complementary to PNA1, but has a mismatch to PNA2. The reverse is true for Codon 112 CGC. By using PNAl as the probe, the DNA involving Codon 112 TGC, another DNA involving Codon 112 CGC, and 1:l mixture of them (a heterozygous sample) were examined. These three samples were treated with nuclease S1 in the presence of PNA1, and the reaction mixtures were stained with DiSC2(5).As expected, the solution of the first DNA (homozygous Codon 112 TGC) exhibited purple color, and the second one (homozygous Codon 112 CGC) was blue. Significantly, the 1:l mixture of them (heterozygous sample) was thin purple because one of the DNAs was completely resistant to nuclease S1 but the other was digested. The W spectra of these three solutions were notably dif€erent from each other (Figure 1).On the similar treatments of various DNA samples in the presence of the corresponding PNA probes, the colors of the digest solutions were as follows: purple (homozygous full-match), thin purple (heterozygote), and blue (homozygous mismatch). Whether the SNP exists in the DNA or not is clearly distinguishable even in heterozygous DNA sample. The results on genotyping of multiplex systems (simultaneous detection of two SNPs) are presented in Table 2. The colors of digests stained with the dye are almost dependent of coexisting DNA Therefore, the genotypes of DNA samples (at two SNPs sites) could be concretely determined by using four PNA probes. It is indicated that simultaneous detection of multiple SNPs will be achieved still more conveniently by extending this method onto a microanay platform and using a number of different PNA probes. In conclusion, the combination of nuclease and PNA should be straightforward, simple, and robust technology for SNP detection . 0. d  (...truncated)