Restriction fragment length polymorphism: Difference between revisions

Revision as of 16:52, 4 November 2017

Revision as of 02:42, 11 November 2017

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== Analysis (technology) ==

The basic technique for the detection of RFLPs involves fragmenting a sample of DNA by a [[restriction enzyme]], which can recognize and cut DNA wherever a [[recognition sequence|specific]] short [[base pairs|sequence]] occurs, in a process known as a [[restriction digest]]. The resulting DNA fragments are then separated by length through a process known as [[agarose gel electrophoresis]], and transferred to a membrane via the [[Southern blot]] procedure. [[Nucleic acid hybridization|Hybridization]] of the membrane to a labeled [[Hybridization probe|DNA probe]] then determines the length of the fragments which are [[Complementarity (molecular biology)|complementary]] to the probe. An RFLP occurs when the length of a detected fragment varies between individuals. Each fragment length is considered an [[allele]], and can be ticsused in [[Genetics|genetic analysis]].

RFLP analysis may be subdivided into single- (SLP) and multi-locus probe (MLP) paradigms. Usually, the SLP method is preferred over MLP because it is more sensitive, easier to interpret and capable of analyzing mixed-DNA samples.{{Citation needed|date=August 2009}} Moreover, data can be generated even when the DNA is degraded (e.g. when it is found in bone remains.)

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=== Examples ===

There are two common mechanisms by which the size of a particular restriction fragment can vary. In the first schematic, a small segment of the genome is being detected by a DNA probe (thicker line). In allele "A", the genome is cleaved by a restriction enzyme at three nearby sites (triangles), but only the rightmost fragment will be detected by the probe. In allele "a", restriction site 2 has been lost by a [[genetic mutation|mutation]], so the probe now detects the larger fused fragment running from sites 1 to 3. The second diagram shows how this fragment size variation would look on a Southern blot, and how each allele (two per individual) might be inherited in members of a family.

In the third schematic, the probe and restriction enzyme are chosen to detect a region of the genome that includes a [[variable number tandem repeat]] segment (boxes in schematic diagram). In allele "c" there are five repeats in the VNTR, and the probe detects a longer fragment between the two restriction sites. In allele "d" there are only two repeats in the VNTR, so the probe detects a shorter fragment between the same two restriction sites. Other genetic processes, such as [[Genetic insertion|insertions]], [[Genetic deletion|deletions]], [[Chromosomal translocation|translocations]], and [[Chromosomal inversion|inversions]], can also lead to RFLPs.