Analytical and Bioanalytical Chemistry

, Volume 395, Issue 3, pp 669–677 | Cite as

Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

  • Martin DufvaEmail author
  • Jesper Petersen
  • Lena Poulsen


DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to function optimally under one condition only. Microarrays are often burdened with a significant degree of cross-hybridization, because of a poor combination of assay conditions and probe choice. As reviewed here, a number of promising microfluidics-based technologies can provide automatic processing of arrays under different assay conditions. These new array processors provide researchers and assay developers with novel possibilities to construct highly specific DNA arrays even towards regions of DNA greatly varying in G + C content. These array processors are also a powerful development tool for building arrays, because they combine high sample throughput with investigation of optimal assay conditions. The array processors can increase specificity in all DNA microarray assays, e.g. for gene expression, and microRNA and mutation analysis. Increased specificity of the array will also benefit microarray-based loci selection prior to high-throughput sequencing.


Microarray DNA sequencing Nearest neighbor method Genotyping Diagnostics Gradient Fluidics 


  1. 1.
    L. Poulsen, M.J. Soe, D. Snakenborg, L.B. Moller, and M. Dufva, (2008) Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface. Nucleic AcidGoogle Scholar
  2. 2.
    Shi L, Reid LH, Jones WD, Shippy R, Warrington JA, Baker SC et al (2006) The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnol 24:1151–1161CrossRefGoogle Scholar
  3. 3.
    Wang Y, Barbacioru C, Hyland F, Xiao W, Hunkapiller KL, Blake J, Chan F, Gonzalez C, Zhang L, Samaha RR (2006) Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays. BMC Genomics 7:59CrossRefGoogle Scholar
  4. 4.
    Albert TJ, Molla MN, Muzny DM, Nazareth L, Wheeler D, Song X, Richmond TA, Middle CM, Rodesch MJ, Packard CJ, Weinstock GM, Gibbs RA (2007) Direct selection of human genomic loci by microarray hybridization. Nat Methods 4:903–905CrossRefGoogle Scholar
  5. 5.
    Hughes TR, Mao M, Jones AR, Burchard J, Marton MJ, Shannon KW, Lefkowitz SM, Ziman M, Schelter JM, Meyer MR, Kobayashi S, Davis C, Dai H, He YD, Stephaniants SB, Cavet G, Walker WL, West A, Coffey E, Shoemaker DD, Stoughton R, Blanchard AP, Friend SH, Linsley PS (2001) Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol 19:342–347CrossRefGoogle Scholar
  6. 6.
    SantaLucia J Jr (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A 95:1460–1465CrossRefGoogle Scholar
  7. 7.
    SantaLucia J Jr, Hicks D (2004) The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 33:415–440CrossRefGoogle Scholar
  8. 8.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415CrossRefGoogle Scholar
  9. 9.
    Wernersson R, Nielsen HB (2005) OligoWiz 2.0--integrating sequence feature annotation into the design of microarray probes. Nucleic Acids Res 33:W611–615CrossRefGoogle Scholar
  10. 10.
    Xu J, Craig SL (2005) Thermodynamics of DNA hybridization on gold nanoparticles. J Am Chem Soc 127:13227–13231CrossRefGoogle Scholar
  11. 11.
    Dufva M, Petersen J, Stoltenborg M, Birgens H, Christensen CB (2006) Detection of mutations using microarrays of poly(C) 10-poly(T) 10 modified DNA probes immobilized on agarose films. Anal Biochem 352:188–197CrossRefGoogle Scholar
  12. 12.
    Fotin AV, Drobyshev AL, Proudnikov DY, Perov AN, Mirzabekov AD (1998) Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips. Nucleic Acids Res 26:1515–1521CrossRefGoogle Scholar
  13. 13.
    Kajiyama T, Miyahara Y, Kricka LJ, Wilding P, Graves DJ, Surrey S, Fortina P (2003) Genotyping on a thermal gradient DNA chip. Genome Res 13:467–475CrossRefGoogle Scholar
  14. 14.
    Petersen J, Poulsen L, Petronis S, Birgens H, Dufva M (2008) Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays. Nucleic Acids Res 36:e10CrossRefGoogle Scholar
  15. 15.
    Vainrub A, Pettitt BM (2003) Surface electrostatic effects in oligonucleotide microarrays: control and optimization of binding thermodynamics. Biopolymers 68:265–270CrossRefGoogle Scholar
  16. 16.
    Vainrub A, Pettitt BM (2002) Coulomb blockage of hybridization in two-dimensional DNA arrays. Phys Rev E Stat Nonlin Soft Matter Phys 66:041905Google Scholar
  17. 17.
    Fixe F, Dufva M, Telleman P, Christensen CB (2004) Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays. Nucleic Acids Res 32:e9CrossRefGoogle Scholar
  18. 18.
    Shchepinov MS, Case-Green SC, Southern EM (1997) Steric factors influencing hybridisation of nucleic acids to oligonucleotide arrays. Nucleic Acids Res 25:1155–1161CrossRefGoogle Scholar
  19. 19.
    Peterson AW, Wolf LK, Georgiadis RM (2002) Hybridization of mismatched or partially matched DNA at surfaces. J Am Chem Soc 124:14601–14607CrossRefGoogle Scholar
  20. 20.
    Halperin A, Buhot A, Zhulina EB (2006) Hybridization at a surface: the role of spacers in DNA microarrays. Langmuir 22:11290–11304CrossRefGoogle Scholar
  21. 21.
    Yao D, Kim J, Yu F, Nielsen PE, Sinner EK, Knoll W (2005) Surface density dependence of PCR amplicon hybridization on PNA/DNA probe layers. Biophys J 88:2745–2751CrossRefGoogle Scholar
  22. 22.
    Bishop J, Blair S, Chagovetz AM (2006) A competitive kinetic model of nucleic acid surface hybridization in the presence of point mutants. Biophys J 90:831–840CrossRefGoogle Scholar
  23. 23.
    Pullat J, Fleischer R, Becker N, Beier M, Metspalu A, Hoheisel JD (2007) Optimization of candidate-gene SNP-genotyping by flexible oligonucleotide microarrays; analyzing variations in immune regulator genes of hay-fever samples. BMC Genomics 8:282CrossRefGoogle Scholar
  24. 24.
    Marcy Y, Cousin PY, Rattier M, Cerovic G, Escalier G, Bena G, Gueron M, McDonagh L, le Boulaire F, Benisty H, Weisbuch C, Avarre JC (2008) Innovative integrated system for real-time measurement of hybridization and melting on standard format microarrays. Biotechniques 44:913–920CrossRefGoogle Scholar
  25. 25.
    Howell WM, Jobs M, Gyllensten U, Brookes AJ (1999) Dynamic allele-specific hybridization. A new method for scoring single nucleotide polymorphisms. Nat Biotechnol 17:87–88CrossRefGoogle Scholar
  26. 26.
    Lee HH, Smoot J, McMurray Z, Stahl DA, Yager P (2006) Recirculating flow accelerates DNA microarray hybridization in a microfluidic device. Lab Chip 6:1163–1170CrossRefGoogle Scholar
  27. 27.
    Noerholm M, Bruus H, Jakobsen MH, Telleman P, Ramsing NB (2004) Polymer microfluidic chip for online monitoring of microarray hybridizations. Lab Chip 4:28–37CrossRefGoogle Scholar
  28. 28.
    Yershov G, Barsky V, Belgovskiy A, Kirillov E, Kreindlin E, Ivanov I, Parinov S, Guschin D, Drobishev A, Dubiley S, Mirzabekov A (1996) DNA analysis and diagnostics on oligonucleotide microchips. Proc Natl Acad Sci U S A 93:4913–4918CrossRefGoogle Scholar
  29. 29.
    Chagovetz A, Blair S (2009) Real-time DNA microarrays: reality check. Biochem Soc Trans 37:471–475CrossRefGoogle Scholar
  30. 30.
    Jobs M, Howell WM, Stromqvist L, Mayr T, Brookes AJ (2003) DASH-2: flexible, low-cost, and high-throughput SNP genotyping by dynamic allele-specific hybridization on membrane arrays. Genome Res 13:916–924CrossRefGoogle Scholar
  31. 31.
    Mao H, Holden MA, You M, Cremer PS (2002) Reusable platforms for high-throughput on-chip temperature gradient assays. Anal Chem 74:5071–5075CrossRefGoogle Scholar
  32. 32.
    S. Petronis, D. Gantzhorn, C. CBV, and M. Dufva, (2005) Multithermal DNA Micro-Array Chip for Rapid DNA Melting Temperature Measurement and Advanced SNP Discrimination. MEMS2005.Google Scholar
  33. 33.
    L. Poulsen, M. Søe Jensen, L. Birk Møller, and M. Dufva, (2008) Guidelines for development of DNA microarray based allele-specific hybridization assays.. Submitted.Google Scholar
  34. 34.
    J. Petersen, L. Poulsen, H. Birgens, and M. Dufva, (2009) Microfludic device for creating ionic strength gradients over DNA microarrays for efficient DNA melting studies and assay development. PLoS ONE 4 e4808.Google Scholar
  35. 35.
    A.E. Pozhitkov, R.D. Stedtfeld, S.A. Hashsham, and P.A. Noble, (2007) Revision of the nonequilibrium thermal dissociation and stringent washing approaches for identification of mixed nucleic acid targets by microarrays. Nucleic Acids Res.Google Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.FAST Group, Department of Micro and NanotechnologyTechnical University of DenmarkKongens LyngbyDenmark
  2. 2.Department of Haematology, Herlev HospitalUniversity of CopenhagenHerlevDenmark
  3. 3.Department of Forensic Medicine, Section of Forensic GeneticsUniversity of Copenhagen, Faculty of Health SciencesCopenhagen ØDenmark

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