Division of Functional Genome Analysis


					Functional Genome Analysis (B070) Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580 D-69120 Heidelberg, Germany.

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DNA-Microarray Technology

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FINISHED PROJECT:
Manufacturing DNA-microarrays of high spot homogeneity and reduced background signal from unpurified PCR-products

Diehl et al. (2002) Nucleic Acids Res. 30, e79.
Diehl et al. (2001) Nucleic Acids Res. 29, e38.

For the production of DNA-microarrays from PCR-products, the purification of the DNA-fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur.

We developed a protocol that permits the manufacturing of microarrays from unpurified PCR-products. The presence of primer molecules in the PCR-sample does not increase unspecific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR-products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays that can be made from one batch of amplification products.

By using betaine as an additive to the spotting solution, both the binding efficiency of spotted PCR-products and the homogeneity of the DNA-spots is improved significantly on aminated surfaces such as glass slides coated poly-L-lysine or aminosilane. In addition, unspecific background signal is markedly diminished. Concomitantly, the betaine reduces evaporation from the microtitre dish wells during the arraying procedure.

Subsequent blocking of the chip surface with succinic anhydride was improved in presence of the unpolar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N-methylimidazole. This procedure prevents overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.

Figure. The amount of DNA present before (left bar) and after purification (right bar) of 24 randomly chosen PCR products. On average, 49% of the material was lost during purification.

FINISHED PROJECT:
Quantitative photolithographic synthesis of individually quality-checked DNA microarrays

For DNA-chip analyses, oligonucleotide quality has immense consequences to accuracy, sensitivity and dynamic range. Quality of chips produced by photolithographic in situ synthesis depends critically on the efficiency of photo-deprotection. By means of base-assisted enhancement of this process using 5'-[2-(2-nitrophenyl)-propyloxycarbonyl]-2'-deoxynucleoside phosphoramidites, synthesis yields improved by at least 12% per condensation compared to current chemistries. Thus, the eventual total yield of full-length oligonucleotide is increased more than tenfold in case of 20-mers. Furthermore, the quality of every individual array position was checked quantitatively after synthesis. Subsequently, the very, quality-tested chips were used in successive hybridisation experiments.

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Beier and Hoheisel (1999) Nucleic Acids Res. 27, 1970-1977.
Beier and Hoheisel (2000) Nucleic Acids Res. 28, e11.
Beier and Hoheisel (2005) Curr. Protocols Nucleic Acids Chem., unit 12.5.
other publications and patents

FINISHED PROJECT:
Photolabile 5'-O-phosphoramidites for the photolithographic production of microarrays of inversely oriented oligonucleotides

Photolabile 3'-O-[2-(2-nitrophenyl)propoxycarbonyl]-protected 5'-phosphoramidites were synthesised for an alternative mode of light-directed production of oligonucleotide arrays. Because of the characteristics of these monomeric building blocks, photolithographic in situ DNA-synthesis occurs in 5'-3' direction, conform to the orientation of enzymatic synthesis. The production of such oligonucleotide chips adds new procedural avenues to the growing number of applications of DNA-microarrays.

Beier et al. (2001) Hel. Chim. Acta 84, 2089-2095.
Beier and Hoheisel (2002) J. Biotechnol. 94, 15-22.
Beier and Hoheisel (2004) Curr. Protocols Nucl. Acids Chem., Unit 12.3.
Beier and Hoheisel (2004) Curr. Protocols Nucl. Acids Chem., Unit 12.4.

.other publications and patents

FINISHED PROJECT:
Label-free and amplification-free detection of microarray analyses by means of peptide nucleic acids (PNAs)

Important aspects in microarray analyses are the sensitivity and selectivity of the binding of the assayed DNA molecules. Since the studied DNA-samples usually require a (PCR) amplification and (fluorescence) labelling prior to analysis, time-consuming and costly preparative steps are required, which also might introduce experimental biases. The structural difference between PNA – used as probe on the array – and a DNA-target permits a direct detection of the nucleic acid by mass spectrometry, a process that is much more sensitive than current detection techniques. Thereby, all the preparative steps could be avoided. Upon hybridisation of a DNA or RNA sample to a PNA-array, the phosphates of the nucleid acids can be utilised as an intrinsic label for detection by secondary ion mass spectrometry (SIMS); PNA molecules are lacking phosphate groups entirely. In collaboration with Heinrich Arlinghaus (University of Münster), we were establising the processes for analysing genomic DNA directly without the need for amplification or labelling.

Label-free detection by TOF-SIMS analysis. A primary ion beam hits the surface, from which secondary ions, including phosphate fragments, are released. PNA does not contain phosphates. Therefore, phosphate ionsare visible in the mass spectrum of a PNA-microarray only upon hybridisation of nucleic acids.

SNP-typing unlabelled DNA. A dilution series of two different PNA oligomers was spotted left to right (spot diameter: 300 µm) in columns of eight copies on a silicon wafer with gold-surface. Hybridisation was with an unlabelled DNA that was complementary to only the PNA on the right. Analysis was by TOF-SIMS. The image is a false-colour representation of the signal intensities at a mass of 79 Da.

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Weiler et al. (1997) Nucleic Acids Res. 25, 2792-2799.
Matysiak et al. (1999) Nucl. & Nucl. 18, 1289-1291.
Matysiak et al. (2001) BioTechniques 31, 896-904.
Brandt et al. (2003) Nucleic Acids Res. 31, e119.
Bauer et al. (2003) Comp. Funct. Genom. 4, 520-524.
Jacob et al. (2003) Peptide Nucleic Acids (Nielsen, P.E., ed.), 261-279.
Jacob et al. (2004) Methods in Mol. Biol. (Niemeyer, C., ed.), 283-294.
Arlinghaus et al. (2004) Appl. Surface Sci. 231-232, 392-396.


Brandt & Hoheisel (2004) Trends Biotechnol. 22, 617-622.
Hellweg et al. (2006) Appl. Surface Sci. 252, 6742-6745.
Brandt et al. (2006) Appl. Surface Sci. 252, 6935-6940.
Jacob et al. (2006) Encyclop. Ref. Genomics Proteomics Mol. Med. (Ganten, D. & Ruckpaul, K., eds.), 1422-1425.
Mraheil et al. (2010) Microb. Biotechnol. 3, 634-657.
----------------------------------------------------------------- other publications and patents

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