Microarray

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Microarray is a collective term for modern molecular biological examination systems that allow the parallel analysis of several thousand individual items in a small amount of biological sample material. There are different forms of microarrays, which are sometimes also referred to as “gene chips” or “ biochips ” because, like a computer chip, they can contain a lot of information in a very small space.

Cdnaarray.jpg

DNA microarrays

A DNA microarray is printed by a robot.

DNA microarrays are used in genome analysis , diagnostics and in investigations into differential gene expression . DNA microarrays are used to detect the amount of mRNA and ncRNA of certain genes or rRNA of certain organisms. There are mainly two different types of DNA microarrays, on the one hand those in which cDNA , oligonucleotides or fragments of PCR products corresponding to the mRNA and ncRNA are printed on the carrier material ("spotted microarrays") and those in which synthetic manufactured oligonucleotides are based ("oligonucleotide microarrays"). These serve as probes that are attached to defined positions on a grid, e.g. B. be applied to glass carriers.

  • The NAPPA , English for nucleic acid programmable protein array , is used for the rapid production of proteins. For this, DNA is printed in high density on arrays and then immersed in a reaction buffer. Proteins formed are then captured by anchors, so-called halo-tag ligands . This process is known as HaloTag-NAPPA. It was developed by the Department of Systems Biology of Plants at TUM together with scientists from the USA and Japan, and published in June 2016.

Protein microarrays

The protein -Microarray as well as a DNA microarray includes a plurality of test fields in confined spaces. However, with the protein microarray, small amounts of protein are fixed on the carrier material in each test field - also known as a spot. The process known as mocking requires a high level of precision because of the small test areas at a short distance and is therefore carried out by special devices.

Either a purified protein, for example an antibody, or a protein mix of the tested sample can now be applied to the array. Those spots in which no interaction takes place remain empty after a washing step has been carried out. The detection method then allows the distinction between spots with and without protein-protein interaction. Quantitative detection methods are also possible in which the amount of adhering protein can be determined.

Types of protein microarrays

Microarrays

The different types of protein microarrays can be differentiated according to the type of interaction ( antigen - antibody , enzyme- substrate, receptor- protein or general protein-protein interaction). It can also be differentiated whether the proteins of the sample are fixed on the array and then tested with a large number of specific, known test proteins - or whether the test proteins are fixed in the test areas and then the reaction with the sample proteins takes place.

  • The reverse phase protein microarray method (also called lysate microarray) is used to detect antigens in cell lysates of different tissues or in protein fractions obtained by isoelectric focusing . The cell lysate or protein fraction is spotted on the carrier material of the microarray, after which the antibody is applied. The antibody adheres to every test field with antibody-antigen interaction. Fields with antibodies can then be detected as with Western blot . This is usually done via a labeled second antibody that binds the antigen-specific first antibody. This second antibody is then coupled with a fluorescent or near infrared dye and is detected with an appropriate scanner, or is coupled with an enzyme, the horseradish peroxidase, which allows a light-emitting reaction or a color reaction (use of chromogens) for detection. Lysate microarrays allow the detection and quantification of an antigen in many different lysates at the same time. This method is limited only by the limited number of specific antibodies required for the precise detection of a specific antigen.
  • Antibody microarrays : The antibodies are fixed (spotted) and then the sample (e.g. complex cell lysates) is applied to the array. The antigen binds to the respective immobilized antibody (so-called capture antibody). These captured antigens must now be detected with a second specific antibody (detection antibody), which is then either labeled itself or is detected with a labeled second antibody. This complex is then detected and quantified using the label (cf. ELISA ).
  • Antigen microarrays : A different antigen is fixed on each test area of ​​the array. If the serum of a blood sample contains the corresponding specific antibody, it will stick to the test area. This enables the reaction to a large number of bacterial antigens or allergens to be tested simultaneously. The first antibody is bound by a labeled second antibody in a further incubation step and can be detected.
  • In protein domain microarrays , fusion proteins are fixed on the array in order to detect protein-protein interactions. The fusion protein enables reliable fixation on the array with the first part without disturbing the ability of the other protein part to interact. The applied protein only sticks to those test areas where there is an interaction.
  • A peptide microarray contains short peptide sequences which, depending on the method, are either synthesized in situ or applied directly to the surface using a laser printer and solid-phase synthesis . This method has several advantages, including: a. lower synthesis costs and a larger number of peptides that can be printed in parallel. Peptide microarrays are u. a. used for profiling enzymes , for examining antibody epitopes ( epitope mapping ), or to clarify the amino acids that are necessary for protein binding. In practice, peptide microarrays u. a. Used for monitoring therapeutic interventions, stratifying patients, profiling immune responses of individual patients as the disease progresses, or for developing diagnostic and therapeutic agents and vaccinations .

One possible advantage over DNA microarrays is the faster on-site analysis of samples, as the often necessary amplification of genetic material and hybridization can be dispensed with. Protein microarrays also allow high-throughput analysis of the protein level. The latest research suggests that mRNA and protein levels do not always correlate. Thus, cDNA microarray results do not necessarily indicate protein expression.

Transfection microarrays

This is a technique in which DNA is applied to the array along with a transfection reagent (alternatively, the array can also be treated with the transfection reagent after spotting). Different cell lines can be cultivated on an array prepared in this way (see cell culture ), which, depending on where on the array they adhere to the surface, are transfected with the respective gene. In this way, many genes can be examined in parallel for the relationship between gene and phenotype in a high-throughput process. This will probably close the gap between genome research and medical diagnostics in the future.

Tissue microarrays

With tissue microarrays (TMA), punched tissue cylinders of different origins are assembled on a paraffin block. Depending on the size of the punch, usually between 0.6 mm and 2 mm in diameter, between 50 and 400 samples can be accommodated on a 1.5 × 3 cm area and simultaneously e.g. B. be examined by means of immunohistology . With this method, for example, numerous samples (e.g. tumors of different origins) can be examined on a slide with just a single application of an antibody. The advantage here is the low material consumption with a large number of data records obtained at the same time. It can be disadvantageous that the punched out tissue section is not representative of the entire tissue. However, this disadvantage usually only arises in so-called complex tissues (e.g. liver). In the usual application with tumor material, this problem is negligible, since in the application of TMA it is not the individual result that matters, but the results of the collective examination. In addition to use in immunohistology, analyzes using in situ hybridization are also possible (FISH, CISH).

Carbohydrate microarrays

Sugar molecules can now also be detected using microarray technology.

history

Microarray technology did not emerge until the 1990s . However, due to the high number of tests per unit of time, the comparatively small amount of samples and the ease with which it can be automated, it quickly established itself as an important component in research in the fields of pharmacy , medicine , biochemistry , biotechnology , genetics and molecular biology .

Before that, gel- based electrophoretic or chromatographic methods were used in these research fields for the same task , which were much more time-consuming. A previous method is the dot-blot analysis.

For protein microarrays, Ekins described in the late 1980s that “microspot assays” are extremely sensitive to detection. Similar approaches have already been described for the production of antibody macroarrays. By the year 2000, the device developments for genome research enabled the production of protein microarrays with many thousands of DNA probes on a very small area.

See also

literature

  • Hans-Joachim Müller, Thomas Röder: The experimenter: microarrays . Spectrum Akademischer Verlag, Heidelberg 2004, ISBN 3-8274-1438-5 .
  • Carolyn R Cho, Mark Labow, Mischa Reinhardt, Jan van Oostrum, Manuel Peitsch: The application of systems biology to drug discovery. In: Current Opinion in Chemical Biology. 10, 2006, pp. 294-302.
  • J. Packeisen, E. Korsching, H. Herbst, W. Boecker, H. Buerger: Demystified ... tissue microarray technology. In: Mol Pathol. 56 (4), 2003 Aug, pp. 198-204.
  • JH Malone, B. Oliver: Microarrays, deep sequencing and the true measure of the transcriptome. In: BMC Biology. 9, 2011, p. 34. (Review) doi: 10.1186 / 1741-7007-9-34

Web links

Individual evidence

  1. ↑ HaloTag NAPPA TU-München
  2. ↑ HaloTag-NAPPA publication
  3. ↑ Volker Stadler, Thomas Felgenhauer, Mario Beyer, Simon Fernandez, Klaus Leibe: Combinatorial Synthesis of Peptide Arrays with a Laser Printer . In: Angewandte Chemie International Edition . tape 47 , no. 37 , September 1, 2008, ISSN  1521-3773 , p. 7132-7135 , doi : 10.1002 / anie.200801616 .
  4. ↑ Frank Breitling, Thomas Felgenhauer, Alexander Nesterov, Volker Lindenstruth, Volker Stadler: Particle-Based Synthesis of Peptide Arrays . In: ChemBioChem . tape 10 , no. 5 , March 23, 2009, ISSN  1439-7633 , p. 803-808 , doi : 10.1002 / cbic.200800735 .