Ligase chain reaction

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The ligase chain reaction (English Ligase Chain Reaction, LCR ) has been a detection method for small amounts of genetic material ( DNA ) since 1989 .

principle

The LCR works in a similar way to the polymerase chain reaction , only with the use of a different enzyme , because a thermostable DNA ligase (e.g. Taq DNA ligase ) is used. Two adjacent hybridization probes are ligated to a primer for each DNA strand . The resulting linked DNA molecules, which are often only 30-50 bp long, serve as a starting point for the ligase in the following cycles. The LCR can be combined with the PCR by adding a thermostable DNA polymerase . The additionally added primers are then used to generate amplificates .

The ligated LCR products can be detected, for example, by measuring ionizing radiation or via enzyme immunoassay (EIA) by marking the 3 'and 5' ends (e.g. fluorescein or biotin ) with different ligands so that only the ligated products are accessible for further detection in the assay. The linked DNA molecules can also be separated and detected by agarose gel electrophoresis or capillary electrophoresis .

Applications

The LCR was experimentally used as a detection system for the detection of bacterial and viral DNA z. B. in the human papillomavirus (Bond 1990, Hampl 1991) or Mycobacterium tuberculosis (Barany 1991) or used for the detection of point mutations using allele-specific primers. The detection threshold for these systems was given as 200–1000 target molecules. The LCR is also used in tumor diagnostics . Compared to PCR with Taq polymerase, the LCR has the advantage that base mismatches are not amplified or even arise from the enzyme activity , so that the LCR can also be run with very high cycle numbers of 50-70 without an increase to observe unspecific products. Therefore the ligase chain reaction is also used for the detection of SNPs .

Individual evidence

  1. M. Wiedmann, WJ Wilson, J. Czajka, J. Luo, F. Barany, CA Batt: Ligase chain reaction (LCR) - review and applications. In: PCR methods and applications. Volume 3, Number 4, February 1994, pp. S51-S64, ISSN  1054-9803 . PMID 8173509 . (PDF).
  2. ^ SC Andras, JB Power, EC Cocking, MR Davey: Strategies for signal amplification in nucleic acid detection. In: Molecular Biotechnology. Volume 19, Number 1, September 2001, pp. 29-44, ISSN  1073-6085 . doi : 10.1385 / MB: 19: 1: 029 . PMID 11697219 .
  3. C. Ong, W. Tai, A. Sarma, SM Opal, AW Artenstein, A. Tripathi: Ligation with nucleic acid sequence-based amplification. In: The Journal of Molecular Diagnostics , Volume 14, Number 3, 2012 May-Jun, pp. 206-213, ISSN  1943-7811 . doi : 10.1016 / j.jmoldx.2012.01.004 . PMID 22449695 . PMC 3349837 (free full text).