International Journal of Oral Biology

The Korean Academy of Oral Biology (대한구강생물학회)
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Surface Polarity Dependent Solid-state Molecular Biological Manipulation with Immobilized DNA on a Gold Surface

  • Lee, Jiyoung (Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Kyung Hee University) ;
  • Kim, Jeong Hee (Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Kyung Hee University)
  • Received : 2012.10.05
  • Accepted : 2012.12.11
  • Published : 2012.12.31
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Abstract

As the demand for large-scale analysis of gene expression using DNA arrays increases, the importance of the surface characterization of DNA arrays has emerged. We compared the efficiency of molecular biological applications on solid-phases with different surface polarities to identify the most optimal conditions. We employed thiol-gold reactions for DNA immobilization on solid surfaces. The surface polarity was controlled by creating a self-assembled monolayer (SAM) of mercaptohexanol or hepthanethiol, which create hydrophilic or hydrophobic surface properties, respectively. A hydrophilic environment was found to be much more favorable to solid-phase molecular biological manipulations. A SAM of mercaptoethanol had the highest affinity to DNA molecules in our experimetns and it showed greater efficiency in terms of DNA hybridization and polymerization. The optimal DNA concentration for immobilization was found to be 0.5 ${\mu}M$. The optimal reaction time for both thiolated DNA and matrix molecules was 10 min and for the polymerase reaction time was 150 min. Under these optimized conditions, molecular biology techniques including DNA hybridization, ligation, polymerization, PCR and multiplex PCR were shown to be feasible in solid-state conditions. We demonstrated from our present analysis the importance of surface polarity in solid-phase molecular biological applications. A hydrophilic SAM generated a far more favorable environment than hydrophobic SAM for solid-state molecular techniques. Our findings suggest that the conditions and methods identified here could be used for DNA-DNA hybridization applications such as DNA chips and for the further development of solid-phase genetic engineering applications that involve DNA-enzyme interactions.

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References

  1. Takahashi J, Misawa M,Iwahashi H. Oligonucleotide microarray analysis of age-related gene expression profiles in miniature pigs. PLoS. One. 2011;6: e19761.
  2. Gorreta F., Carbone W, Barzaghi D. Genomic profiling: cDNA arrays and oligoarrays. Methods Mol Biol. 2012; 823:89-105.
  3. Krichevsky AM, King KS, Donahue CP, Khrapko K, Kosik KS. A microRNA array reveals extensive regulation of microRNAs during brain development. RNA. 2003;9:1274- 1281.
  4. Gerhold D, Rushmore T, Caskey CT. DNA chips: promising toys have become powerful tools. Trends Biochem Sci. 1999;24:168-173.
  5. Mikhailovich VD, Gryadunov A, Kolchinsky A AA, Makarov AA, Zasedatelev, A. DNA microarrays in the clinic: infectious diseases. Bioessays. 2008;30:673-682.
  6. De Leeuw N, Hehir-Kwa JY,Simons A, Geurts van Kessel A, Smeets DF, Faas BH, Pfundt R. SNP array analysis in constitutional and cancer genome diagnostics - copy number variants, genotyping and quality control. Cytogenet Genome Res. 2011;135:212-21
  7. Harakalova M, Mokry M, Hrdlickova B, Renkens I, Duran K, van Roekel H, Lansu N, van Roosmalen M, de Bruijn E, Nijman IJ, Kloosterman WP, Cuppen E. Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing. Nat Protoc. 2011;6:1870-1886.
  8. Gronewold T.M. Aptamers and biosensors. Methods Mol Biol. 2009;535:209-222.
  9. Stoevesandt O, Taussig MJ, He M. Producing protein microarrays from DNA microarrays. Methods Mol Biol. 2011;785:265-276.
  10. Zhang X, Yadavalli VK. Surface immobilization of DNA aptamers for biosensing and protein interaction analysis. Biosens Bioelectron. 2011;26:3142-3147.
  11. Rasmussen S.R, Larsen MR, Rasmussen SE. Covalent immobilization of DNA onto polystyrene microwells: the molecules are only bound at the 5' end. Anal Biochem. 1991;198:138-142.
  12. Herne TM, Tarlov MJ () Characterization of DNA probes immobilized on gold surfaces. J Am Chem Soc. 1997; 119:8916-8920.
  13. Steel AB, Levicky RL, Herne TM, Tarlov MJ. Immobilization of nucleic acids at solid surfaces: effect of oligonucleotide length on layer assembly. Biophys J. 2000;79: 975-981.
  14. Wang J. From DNA biosensors to gene chips. Nucleic. Acids Res.2000;28:3011-3016.
  15. Tombelli S, Mascini M, Turner AP. Improved procedures for immobilisation of oligonucleotides on gold-coated piezoelectric quartz crystals. Biosens Bioelectron. 2002;17:929-936.
  16. Peelen, D., and L.M. Smith (2005) Immobilization of aminemodified oligonucleotides on aldehyde-terminated alkanethiol monolayers on gold. Langmuir. 2005;21:266-271.
  17. Loaiza OA, Campuzano S, Pedrero M, Pingarrón JM. DNA sensor based on an Escherichia coli lac Z gene probe immobilization at self-assembled monolayers-modified gold electrodes. Talanta. 2007;73:838-844.
  18. Kaufmann R, Averbukh I, Naaman R, Daube SS. Controlling the reactivity of adsorbed DNA on template surfaces. Langmuir. 2008;24:927-931.
  19. Lim HI, Oliver PM, Marzillier J, Vezenov DV. Heterobifunctional modification of DNA for conjugation to solid surfaces. Anal Bioanal Chem. 2010;397:1861-1872.
  20. Prime KL, Whitesides GM. Self-assembled monolayers:- model system for studying adsorption of protein at surfaces. Science. 1991;252:1164-1167.
  21. Shchepinov MS, Case-Green SC, Southern EM. Steric factors influencing hybridisation of nucleic acids to oligonucleotide arrays. Nucleic Acids Res. 1997;25:1155-1161.
  22. Watterson JH, Raha S, Kotoris CC, Wust CC, Gharabaghi F, Jantzi SC, Haynes NK, Gendron NH, Krull UJ, Mackenzie AE, Piunno PA. () Rapid detection of single nucleotide polymorphisms associated with spinal muscular atrophy by use of a reusable fibre-optic biosensor. Nucleic Acids Res. 2004;32:e18.
  23. Piunno PA, Krull UJ.() Trends in the development of nucleic acid biosensors for medical diagnostics. Anal Bioanal Chem. 2005;381:1004-1011.
  24. Gresham D. DNA microarray-based mutation discovery and genotyping. Methods Mol Biol. 2011;772:179-191.
  25. Solomun T, Mix R, Sturm H. Immobilization of silanized DNA on glass: influence of the silane tether on the DNA hybridization. ACS Appl. Mater. Interfaces. 2010;2:2171-2174.
  26. Kimura N. One-step immobilization of poly (dT)-modified DNA onto non-modified plastic substrates by UV irradiation for microarrays. Biochem Biophys Res Commun. 2006; 347:477-484.
  27. Choithani J, Vaijayanthi B, Kumar P, Gupta KC. Construction of oligonucleotide microarrays (biochip) using heterobifunctional reagents. Methods Mol Biol. 2007;381:133-163.
  28. Sethi D, Kumar P, Gupta KC. Strategies for preparation of oligonucleotide biochips and their applications. Nucleic Acids Symp. Ser. 2009;53149-150.
  29. Watterson JH, Piunno PA, Wust CC, Raha S, Krull UJ. Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors. Fresenius. J Anal Chem. 2001;369:601-608.
  30. Diehl F, Grahlmann,S, Beier M, Hoheisel, JD. Manufacturing DNA microarrays of high spot homogeneity and reduced background signal. Nucleic Acids Res. 2001;29: E38.