FENXI HUAXUE Chinese Journal of Analytical Chemistry. Single nucleoitide polymorphism SNP. PCR Gold magnetic nanoparticles GMNPs ssdna-gmnps SNP

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1 FENXI HUAXUE Chinese Journal of Analytical Chemistry ~ 1713 DOI /SP. J * Single nucleoitide polymorphism SNP PCR Gold magnetic nanoparticles GMNPs ssdna-gmnps PCR DNA 24 MTHFR C677T 1 SNP ~ 9 10 ~ 13 Lien 12 DNA SNP Nakagawa 13 DNA ALDH2 SNP MTHFR C677T Cy3 Cy Nos No. 2007AA Nos. 2007CB CB No No. 2009ZX No C No. 08B018 No. 09JJ JJ3017 * nyhe1958@ 163. com

2 PTC 220 PCR MJ Research GenePix 4100A Axon Nano-Plotter Gesim 1 MTHFR C677T Table 1 Oligonucleotide sequence 1 Name Type Sequence 5'-3' C677T-F Primer TGAAGGAGAAGGTGTCTGCGGGA Biotin-C677T-R Biotin-labeled primer Biotin- T 15 AGGACGGTGCGGTGAGAGTG C677T-CC Cy3 labeled probes Cy3-CGGGAGCCGATTT Taq DNA Taq DNA C677T-TT Cy5 labeled probes Cy5-CGGGAGTCGATTT polymerase 5U /μl dntp Thermo Fisher Sigma-Aldrich NH 4 Fe SO H 2 O 99. 0% NH 4 Fe SO H 2 O 99. 0% NaOH 95% AuCl 3 HCl 4H 2 O 3-Mercaptopropyltriethoxysilane MPTES Sigma-Aldrich - 36 nm 4. 0 g /L PBS ph EDTA DNA QIAGEN D PCR SA-GMNPs ssdna-gmnps HoW CC Cy3 HoM TT Cy5 Cy3 Cy Gold coated magnetic nanoparticles GMNPs 16 1 mg 200 μg 0. 1 mol /L PBS ph ml 0. 1 mol /L PBS 2 4 g /L PBS PCR DNA PCR 95 dsdna-gmnps ssdna- GMNPs - DNA DNA ssdna C677T PCR SNP PCR MTHFR C677T 30 μl 10 PCR 3 μl 25 mmol /L Mg μl 10 mmol /L dntp 0. 6 μl PCR 1 ml U Taq DNA 5 U /L DNA 80 ng PCR 94 5 min s s s min μg μl 10 μmol /L 5'-Biotin-TGAAGGAGAAGGTGTCTGCGGGA-Cy3 5'-Biotin-

3 TGAAGGAGAAGGTGTCTGCGGGA-Cy5 15 min ddh 2 O 5 μl ddh 2 O μl ss-dna SA-GMNPs 3 SSC ddh 2 O 2 3 min SNP MTHFR C677T PCR 10 μmol /L 677CC 677TT 6. 5 μl 22 μl 30 μl 38 1 h 3 SSC ddh 2 O Cy3 Cy5 4100A Axon USA Cy3 550 nm 570 nm Cy5 649 nm 670 Cy3 Cy5 Genepix 6. 0 Origin SNP SNP 3 I = log Signal Cy3 + Cy5 G = Signal Cy3 /Signal Cy3 + Cy I > 3 I G 1 G 0 G G < 0. 2 HoM < G < He G > 0. 8 HoW I G SNP Fig. 1 Quality detection standard of single nucleotide polymorphism SNP genotyping results 2A 2B 8%

4 DNA PCR 10 μmol /L 1 μl μmol /L 3 MTHFR C677T PCR PCR 3b Fig. 2 Immobility assay of magnetic bead array 1 4 A. Fluorescence images before and after washing B. Relative fluorescence intensities before and after washing. Cy Cy PCR Fig. 3 DNA Effect of primer proportion on SNP genotyping after asymmetry PCR DNA PCR PCR PCR - PCR PCR μmol /L μmol /L DNA S1 ~ S5 S'1 ~ S'5 50% PCR μmol /L 3. 4 SNP SNP 24 C677T 5A C677T B 24 I G SNP I > 3 24

5 C677T G > PCR Cy3 677CC 677CC 3 G < PCR Cy5 677TT 677TT 8 G = ~ Cy3 677CC Cy5 677TT 677CT 4 DNA Fig. 4 Effect of primer concentration on SNP genotyping 95 PCR of five samples after asymmetry PCR A. Preparation of scanning results B. Comparation of relative PCR DNA fluorescence intensities. 5 C677T 24 Fig. 5 Results of SNP detection of 24 samples based on bead array and dual-color hybridization assayed for C677T locus. A Fluorescence images of microarray B All spots were measured for Cy3 and Cy5 fluorescence intensities to generate a scatter plot PCR References 1 Fan J B Chee M S Gunderson K L. Nat. Rev. Genet ~ Pastinen T Raitio M Lindroos K Tainola P Peltonen L Syv nen A C. Genome Res ~ ZHANG Xiao-Yan ZUO Ming-Xue ZHANG Zhan-Jun WANG Zhong LI Yao. China Biotechnology ~ 56 4 Schena M Heller R A Theriault T P Konrad K Lachenmeier E Davis R W. Trends Biotechnology ~ Dieh F Beckmann B Kellner N Hauser N C Diehl S Hoheisel J D. Nucleic Acids Res e79

6 WANG Feng-Hua LIU Jun TONG Chun-Yi WANG Qi-Ming TANG Dong-Ying YI Long WANG Ling-Ling LIU Xuan-Ming. Chinese J. Anal. Chem ~ Saiyed Z M Bochiwal C Gorasia H Telang S D Ramchand C N. Analytic Biochemistry ~ ZHANG Zhi-Chao YUAN Cui WAN Qian-Hong. Chinese J. Anal. Chem ~ 36 9 LIU Ya-Ling JIA Li XING Da. Chinese J. Anal. Chem ~ Zhou H J Xing D Zhu D B Zhou X M. Talanta ~ Yoshino T Tanaka T Takeyama H Matsunaga T. Biosensors and Bioelectronics ~ Lien K Y Liu C J Lin Y C Kuo P L Lee G B. Microfluid Nanofluid ~ Nakagawa T Hashimoto R Maruyama K Tanaka T Takeyama H Matsunaga T. Biotechnol. Bioeng ~ Li S Liu H N Wang Z F Hou P Guo Y F He Q G He N Y. Anal. Biochem ~ Liu H N Li S Wang Z F Ji M J Nie L B He N Y. Journal of Biotechnology ~ Takuya K Satoshi S Yoshiteru M Yohei O Takashi N Kenji O Takao A Y. J. Magn. Magn. Mater ~ 110 Single Nucleotide Polymorphism Genotyping Based on Magnetic Bead Array and Dual-color Hybridization LIU Hong-Na 1 LI Song 1 2 LIU Li-Shang 2 TIAN Lan 1 JIA Ying-Ying 1 LI Zhi-Yang 2 DENG Yan 1 2 HE Nong-Yue * State Key Laboratory of Bioelectronics Southeast University Nanjing Hunan Key Laboratory of Green Packaging and Application of Biological Nanotechnology Hunan University of Technology Zhuzhou Abstract An approach to fabricate magnetic bead array using gold coated magnetic nanoparticles was developed. Single strand DNAs ssdna obtained using asymmetry PCR were captured on gold coated magnetic nanoparticles SA-GMNPs modified with streptavidin. The GMNPs-ssDNA mixtures were spotted onto a clean glass slide adhered with a magnet to fabricate a bead array and then interrogated by hybridization with a pair of dual-color probes to determine single nucleotide polymorphism SNP. The genotype of each sample can be simultaneously identified on the surface of GMNPs by scanning the microarray. The amplification factors were optimized to obtain ssdna directly by use of polymerase chain reaction PCR. In this method the genotyping results were obtained from 24 different samples analyzed for C677T loci. The method described here is simple and easily to be automated which is suitable for molecular diagnostics and forensic analysis. Keywords Magnetic bead array Asymmetry polymerase chain reaction Dual-color hybridization Single nucleotide polymorphism type Received 25 February 2010 accepted 5 June 2010