| Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future. SYSTEMS BIOLOGYThe study of the complex interactions that occur at all levels of biological information -from whole-genome sequence interactions to developmental and biochemical networks -and their functional relationship to organism-level phenotypes. NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 465 and concomitantly reducing the complexity of genomic DNA. Although PCR can be used to amplify small numbers of DNA molecules in low microlitre or nanolitre volumes in vitro, problems are associated with the dilution of heterozygous DNA. This could result in incorrect genotyping results (homozygous genotypes) because of the preferential amplification of one of the alleles. Extreme dilution indicates that there is a sufficiently high integrity of the nucleic-acid templates to allow efficient amplification. Furthermore, amplification of low concentrations of nucleic acids requires strict quality control, as it is often associated with contamination problems, which can be problematic in high-throughput applications. In contrast to nucleic-acid manipulation, it is currently impossible to amplify proteins from a biological sample. R E V I E W S Miniaturization in functional genomicsGenome research and functional genomics have catalysed the development of high-throughput and miniaturized approaches for the analysis of biomolecules [5][6][7][8] . Figure 1 | DNA micorarrays. DNA microarrays that are based on non-porous solid supports such as glass have paved the way for highly parallel miniaturized analysis of biomolecules. Using robotic workstations, ~0.25-1 nl of DNA solution are printed on a slide, creating spots that range from 100 to 150 µm in diameter. Successive samples are then spaced to avoid contact between adjacent spots, with approximately 200 µm between each spot 7 . Currently, about 50,000 cDNAs can be robotically spotted onto a microscope slide and hybridized with a fluorescently labelled probe. Using photolithographical masking techniques, arrays that have 400,000 distinct oligonucleotides have been produced, each in its own 20-µm 2 region (today, this can be as small as ~5 µm 2 ) REF. 8. After spotting, nucleic-acid samples can be hybridized on these pre-structured microarrays. Hybridization events are monitored using fluorescence scanners. A typical fluorescence read-out of highly parallel hybridization on a cDNA microarray is shown in the bottom panel. Box 1 | Technological problems in miniaturizationEvery molecular biological method can be miniaturized from microl...
The expression and characterization of large protein libraries requires high-throughput tools for rapid and cost-effective expression and screening. A promising tool to meet these requirements is miniaturized high-density plates in chip format, consisting of an array of wells with submicroliter volumes. Here, we show the combination of nanowell chip technology and cell-free transcription and translation of proteins. Using piezoelectric dispensers, we transferred proteins into nanowells down to volumes of 100 nL and successfully detected fluorescence using confocal laser scanning. Moreover, we showed cell-free expression of proteins on a nanoliter scale using commercially available coupled transcription and translation systems. To reduce costs, we demonstrated the feasibility of diluting the coupled in vitro transcription and translation mix prior to expression. Additionally, we present an enzymatic inhibition assay in nanowells to anticipate further applications, such as the high-throughput screening of drug candidates or the identification of novel enzymes for biotechnology.
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