Restriction fragment length polymorphism

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Restriction fragment length polymorphism , abbreviated RFLP ( Engl. : Restriction Fragment Length Polymorphism ) is a method for determining the genetic fingerprint . Here are DNA fragments by restriction enzymes cut and by using a gel electrophoresis its length arranged, whereby two different DNA samples can be compared.

principle

For example, a sequence in person 1 contains a cleavage site for a restriction enzyme , but this does not occur in the sequence in person 2. If these sequences are now cut with a restriction enzyme, two fragments are created for person 1 and one fragment for person 2. If the lengths of the sequences are compared, an RFLP can be determined, the fragments are of different lengths, the locus is polymorphic .

properties

The RFLP method is used in forensic science or in paternity tests . It was the first inexpensive method to compare the relationship between two samples, but is increasingly being used by other biochemical methods such as DGGE or TGGE , phospholipid analysis, polymerase chain reaction (partly with DNA sequencing ), RAPD , STR analysis, the SSCP analysis or further developments of the RFLP such as AFLP , T-RFLP, ARISA, ARDRA . Ribotyping is a variant of RFLP with rDNA .

RFLPs are used, among other things. a. as genetic markers in gene mapping , since the closer they are, the more likely they are to be inherited together. They are also used to search for quantitative trait loci , i.e. chromosome segments that influence the expression of a quantitative trait, as well as in Southern blots .

A more specific application is the T-RFLP (terminal RFLP). The target genes are amplified in the polymerase chain reaction (PCR) with one (or both) fluorescence-labeled primer (s). The amplification products are digested with a restriction enzyme and analyzed by an automated sequencer. This only detects the fluorescent fragments (i.e. the terminal ones). This is used, for example, to determine the diversity in a sample or for diversity comparisons along a gradient. Using the T-RFLP, the compositions of microbiomes from different habitats can also be identified and other metagenomic data determined.

literature

  • Bruce Alberts , Alexander Johnson, Peter Walter, Julian Lewis, Martin Raff, Keith Roberts: Molecular Biology of the Cell. 4th edition. Garland, New York NY 2002, ISBN 0-8153-3218-1 .

Individual evidence

  1. R. Rudner, B. Studamire, ED Jarvis: Determinations of restriction fragment length polymorphism in bacteria using ribosomal RNA genes. In: Methods in Enzymology . Volume 235, 1994, pp. 184-196, ISSN  0076-6879 . PMID 7520118 .
  2. TL Marsh: Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. In: Current Opinion in Microbiology. Volume 2, Number 3, June 1999, pp. 323-327, ISSN  1369-5274 . doi : 10.1016 / S1369-5274 (99) 80056-3 . PMID 10383864 .
  3. IC Anderson, JW Cairney: Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. In: Environmental microbiology. Volume 6, Number 8, August 2004, pp. 769-779, ISSN  1462-2912 . doi : 10.1111 / j.1462-2920.2004.00675.x . PMID 15250879 .
  4. Amélia Camarinha-Silva, Melissa L. Wos-Oxley, Ruy Jáuregui, Karsten Becker, Dietmar H. Pieper: Validating T-RFLP as a sensitive and high-throughput approach to assess bacterial diversity patterns in human anterior nares. FEMS Microbiology Ecology 79 (1) January 2012; Pp. 98-108. doi: 10.1111 / j.1574-6941.2011.01197.x. , Full text .