Colony polymerase chain reaction

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The colony polymerase chain reaction ( English colony PCR ) is a molecular biological and biochemical method for the detection of certain DNA sequences in colonies of bacteria or fungi using a variant of a polymerase chain reaction .

principle

The colony polymerase chain reaction does not use purified plasmid DNA or chromosomal DNA as a DNA template , but rather bacterial colonies removed from the culture medium . In the case of plasmids with a high number of copies, just a few cells are sufficient, but they must be brought into suspension in the PCR approach. Due to the binding of magnesium ions from the PCR buffer by the bacteria and due to the increase in the synthesis rate of the thermostable DNA polymerase (see PCR optimization ), the magnesium concentration is increased to 5 millimolar compared to the PCR and occasionally the chaotrope dimethyl sulfoxide is added. The lysis of the cells and release the DNA from the cytoplasm is effected either by a previous lysis by brief heating to 95 ° C and a purification or by additives in the buffer, such as EDTA or SDS in combination with the heating step at the start of PCR or without Pretreatment with a lysis in the first denaturation step of the PCR. In yeast , a colony polymerase chain reaction with a lysis-buffer with PCR was Lytikase , Octoxinolen , polysorbates and gelatin described. A variant of the colony polymerase chain reaction with reverse transcription is used for single cell analysis of mammalian cells. After the PCR, the amplified DNA is usually analyzed by agarose gel electrophoresis , and occasionally DNA sequencing .

Since components of the culture medium (both liquid and solid media) can inhibit the reaction, their amount is kept as low as possible. In the colony polymerase chain reaction, a positive control and a negative control are mostly used in parallel . The positive control contains the same DNA (occasionally with a different length of the transgene ) as well as the same primer and shows the basic functionality of the reaction. In contrast, the negative control (mostly without adding DNA) indicates contamination . Since no purified DNA is used, the presence of genomic DNA and cellular proteins can lead to incorrect binding of the primer and thus to an increased number of false positive products of the PCR. However, the false positive products usually have a different length than the desired product and can then be differentiated in an agarose gel electrophoresis.

Applications

Colony PCR is a fast variant of the polymerase chain reaction, as colonies of the microorganisms are used directly. It is used to check the successful transformation of DNA into the cells of the bacteria. It is also used to identify recombinant plasmids. Another area of ​​application is taxonomy , in order to investigate the relationships between species or genera through phylogenetic studies . The colony polymerase chain reaction is also used in the diagnosis of pathogens in human and veterinary medicine .

literature

  • Cornel Mülhardt: The Experimenter: Molecular Biology / Genomics. Sixth edition. Spectrum Akademischer Verlag, Heidelberg 2008, ISBN 3-8274-2036-9 .
  • Bing-Yuan Chen, Harry W. Janes: PCR Cloning Protocols . In: Methods in Molecular Biology , Volume 192 Springer 2002. ISBN 9781592591770 . P. 130.
  • AR Pavlov, NV Pavlova, SA Kozyavkin, AI Slesarev: Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes. In: J. Kieleczawa: DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett 2006. ISBN 0-7637-3383-0 . Pp. 241-257.

Web links

  • Cornel Mülhardt: Colony PCR . In: Laborjournal online . Retrieved January 6, 2014.

Individual evidence

  1. ^ MA Hofmann, DA Brian: Sequencing PCR DNA amplified directly from a bacterial colony. In: BioTechniques. Volume 11, Number 1, July 1991, pp. 30-31, ISSN  0736-6205 . PMID 1954013 .
  2. ^ AC Ward: Rapid analysis of yeast transformants using colony PCR. In: BioTechniques. Volume 13, Number 3, September 1992, p. 350, ISSN  0736-6205 . PMID 1389166 .
  3. ↑ a b M. Bergkessel, C. Guthrie: Colony PCR. In: Methods in enzymology. Volume 529, 2013, pp. 299-309, ISSN  1557-7988 . doi : 10.1016 / B978-0-12-418687-3.00025-2 . PMID 24011056 .
  4. ↑ a b H. Packeiser, C. Lim, B. Balagurunathan, J. Wu, H. Zhao: An extremely simple and effective colony PCR procedure for bacteria, yeasts, and microalgae. In: Applied biochemistry and biotechnology. Volume 169, Number 2, January 2013, pp. 695-700, ISSN  1559-0291 . doi : 10.1007 / s12010-012-0043-8 . PMID 23271627 .
  5. ↑ a b c Tapan Ganguly, Peiqin Chen, Rebecca Teetsel, Lan Ping Zhang, Elias Papaioannou, Joseph Cianciarulo: High-throughput sequencing of high copy number plasmids from bacterial cultures by heat lysis. In: Biotechniques (2005), Volume 39, Number 3, pp. 304-308. PDF ( Memento of the original from February 22, 2014 in the Internet Archive ) Info: The archive link was automatically inserted and not yet checked. Please check the original and archive link according to the instructions and then remove this notice. . @1@ 2Template: Webachiv / IABot / www.biotechniques.com
  6. ↑ TJ Kwiatkowski, HY Zoghbi, SA Ledbetter, KA Ellison, AC Chinault: Rapid identification of yeast artificial chromosome clones by matrix pooling and crude lysate PCR. In: Nucleic acids research. Volume 18, Number 23, December 1990, pp. 7191-7192, ISSN  0305-1048 . PMID 2263507 . PMC 332835 (free full text).
  7. ↑ A. Ståhlberg, M. Kubista: The workflow of single-cell expression profiling using quantitative real-time PCR. In: Expert review of molecular diagnostics. Volume 14, Number 3, April 2014, pp. 323-331, ISSN  1744-8352 . doi : 10.1586 / 14737159.2014.901154 . PMID 24649819 . PDF .
  8. ↑ V. Cattoir, O. Lemenand u. a .: The sodA gene as a target for phylogenetic dissection of the genus Haemophilus and accurate identification of human clinical isolates. In: International journal of medical microbiology: IJMM. Volume 296, No. 8, December 2006, pp. 531-540, ISSN  1438-4221 . doi : 10.1016 / j.ijmm.2006.06.005 . PMID 17049306 .
  9. ↑ JR Hayes, DD Wagner a. a .: Distribution of streptogramin resistance determinants among Enterococcus faecium from a poultry production environment of the USA. In: The Journal of antimicrobial chemotherapy. Vol. 55, No. 1, January 2005, pp. 123-126, ISSN  0305-7453 . doi : 10.1093 / jac / dkh491 . PMID 15574480 .