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  • Review Article
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Miniaturization in functional genomics and proteomics

Nature Reviews Genetics volume 6, pages 465–476 (2005)Cite this article

Key Points

  • Large biological data sets provide the basis for systems biology. In this article, we present a selection of miniaturized methods for the analysis of nucleic acids and proteins, with a particular focus on in vitro methods.

  • The reduction of complexity is, in many cases, an important pre-requisite for studying heterogeneous biological samples.

  • High-throughput generation of molecular data can be achieved through parallelization and miniaturization of biological assays. The development of DNA microarrays was one of the key steps in functional genomics, and has been continued with the introduction of protein microarrays.

  • New highly specific and sensitive (single-molecule) approaches for the detection of nucleic acids and proteins can be useful for diagnostic applications.

  • In this review, we mainly discuss three general miniaturization formats: microarrays, nanowell plates and microchannels. Integration of miniaturized systems can represent a major hurdle for high-throughput application.

  • Fluorescence labelling or detection methods that are based on mass spectrometry and electrochemistry are currently mainly being used in biology. Although fluorescence and mass spectrometry procedures are complementary approaches, particularly in basic research, electrical detection is an emerging tool in diagnostics.

  • Agreements on data standardization and interconnected databases are important for consistent analysis of different biological data sets.

Abstract

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.

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Figure 1: DNA micorarrays.
Figure 2: Nanowell plates.
Figure 3: Protein microarrays.
Figure 4: Identification of protein–DNA interactions using the ChIP-chip approach.
Figure 5: The principle of MALDI imaging.

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Acknowledgements

We would like to thank W. Nietfeld for providing Fig. 1 and would like to acknowledge the European Union, the Senatsverwaltung für Wissenschaft, Forschung und Kultur, Berlin (Ultra-Structure Network), the German Ministry for Research and Education (NGFN), and the Max Planck Society for financial support.

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Authors and Affiliations

  1. Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Ihnestrasse 73, Berlin, D-14195, Germany

    Sascha Sauer, Bodo M. H. Lange, Johan Gobom, Lajos Nyarsik, Harald Seitz & Hans Lehrach

Authors
  1. Sascha Sauer
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  2. Bodo M. H. Lange
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  3. Johan Gobom
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  4. Lajos Nyarsik
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  5. Harald Seitz
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Correspondence to Sascha Sauer.

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FURTHER INFORMATION

Biochemical Tools Group at the Max Planck Institute

Bruker Daltonics web site

Ensembl

GenomeMatrix Database

International HapMap Project

Hans Lehrach's homepage

Max Planck Institute for Molecular Genetics

Microarray Gene Expression Data Society web site

MolTools (Advanced Molecular Tools for Array-based Analyses of Genomes)

Protein Structure Factory

RZPD — German Resource Centre for Genome Research

Glossary

SYSTEMS BIOLOGY

The 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.

LASER CAPTURE MICRODISSECTION

A method in which cells are cut out from a tissue sample using a laser beam.

CELL FRACTIONATION

Separation of cells by size, weight, density, and optical and bioelectrical properties.

MONOLITHIC SUBSTRATES

A substrate made from silicon or polymers that allows microfabrication of planar fluidic devices that have integrated microchannels or functional elements such as electrodes and detectors.

MICROSATELLITE

A class of repetitive DNA sequences that are made up of tandemly organized repeats that are 2–8 nucleotides in length. They can be highly polymorphic and are frequently used as molecular markers in population genetics studies.

DNA MICROARRAY

An array of PCR products or oligonucleotides (corresponding to either genomic or cDNA sequences) that are deposited on solid glass slides and can be used to interrogate complex nucleic-acid samples by hybridization.

MASS SPECTROMETRY

An analytical technique for the determination of molecular mass. Although they vary greatly in design, all mass spectrometers share three general components: an ion source in which gas-phase molecular ions are produced from the analyte molecules, a mass analyser in which electrical and/or magnetic fields are used to separate the analyte ions by their different mass-to-charge (m/z) ratios and a detector for recording the separated ions.

HAPLOTYPE

The combination of alleles or genetic markers that is found on a single chromosome of a given individual.

ROLLING-CIRCLE AMPLIFICATION

A mode of DNA replication used by circular DNAs, which generates molecules that look similar to lariats. It was traditionally associated with certain bacterial plasmids and viruses, but is increasingly used as an alternative method for DNA amplification.

TILING OLIGONUCLEOTIDE MICROARRAYS

These microarrays contain a set of overlapping oligonucleotides that span either the entire genome, or for a more specialized approach, a subregion of interest.

LASER SCANNING CONFOCAL MICROSCOPE

A light microscope that allows imaging of fluorescent structures in thick (tens to hundreds of micrometres) specimens. A series of optical 'slices' are collected using a scanning laser beam and specially designed optics to eliminate out-of-focus excited fluorescence. The slices are reconstructed to provide detailed three-dimensional representations of the image data.

X-RAY CRYSTALLOGRAPHY

The study of the molecular structure of crystalline compounds through X-ray diffraction techniques. When an X-ray beam bombards a crystal, the atomic structure of the crystal causes the beam to scatter (diffract) in a specific pattern. X-ray crystallography provides information on the positions of individual atoms in the crystal, the distances between them, the angles of the atomic bonds and other features of molecular geometry.

CRYO-ELECTRON MICROSCOPY

Specimens are quick-frozen by plunging them into liquid ethane and are then kept frozen throughout the imaging process. Rapid freezing causes the water to form vitreous ice around the sample, preserving its native structure. Analysis of the structure can then be carried out in this state by electron microscopy.

MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY (MALDI MS).

An ionization technique that is suitable for mass spectrometric analysis of large biomolecules. Samples are prepared by embedding analyte molecules in a condensed matrix of small molecules. A brief laser pulse irradiates a spot on the sample, resulting in ablation of a small volume of the matrix and desorption of the embedded analyte molecules. Analyte molecules are then ionized, which leads to the formation of predominantly singly charged positive and negative analyte ions.

YEAST TWO-HYBRID (Y2H) ASSAY

One protein is fused to a transcriptional activation domain (the GAL4 activation domain) and the other to a DNA-binding domain (the GAL4 DNA-binding domain), and both fusion proteins are introduced into yeast. Expression of a (GAL4-regulated) reporter gene with the appropriate DNA-binding sites upstream of the promoter indicates that the two proteins physically interact.

TANDEM AFFINITY PURIFICATION

A method that uses the expression of fusion proteins in cells carrying a double tag that is applied in two consecutive steps of purification. This protocol has the benefit of high levels of purification and native protein elution for subsequent functional, structural or biochemical analysis.

ELECTROMOBILITY-SHIFT ASSAY

An assay in which proteins that bind to a DNA fragment are detected by virtue of their reduced migration in an electrical field. The assay is often used to detect transcription-factor binding.

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA).

A widely used immunochemical method for detecting antigens or antibodies. ELISA methods are carried out in microtitre plates and use colorimetric detection.

PEPTIDE NUCLEIC ACID

A biopolymer molecule that consists of DNA bases connected by a backbone of peptide bonds, rather than phosphodiester bonds as in natural DNA.

SIZE-EXCLUSION CHROMATOGRAPHY

This method makes use of the different chromatographical behaviour that depends on the size of molecules and partly on their shape. The extent of the size exclusion of molecules is determined by the steric hindrance of analyte molecules that is due to matrix substance. Small molecules can more or less access the interior of the matrix, whereas large molecules pass by the matrix.

ELECTROSPRAY IONIZATION

An ionization technique that is suitable for mass spectrometric analysis of large biomolecules. The sample, dissolved in an aqueous-organic solvent, is pumped through a fine capillary that is made of electrically conductive material. The high voltage that is applied results in the emission of an aerosol of charged droplets of the analyte solution. Using heated gas, the spray is directed through a series of chambers that have successively decreased pressures, which results in the formation of gas-phase analyte ions.

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Sauer, S., Lange, B., Gobom, J. et al. Miniaturization in functional genomics and proteomics. Nat Rev Genet 6, 465–476 (2005). https://doi.org/10.1038/nrg1618

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