(77)
8. Beskrive begreberne Sothern Blotting, Restriction Fragment Lenght Polymorphism (RFLP) og Singe-Strand Conformational Polymorphism (SSCP)
Stryer, s. 146, fig. 6.3
Devlin, s.297-298, fig. 7.13

Southern Blotting

This technique makes it possible to identify a particular DNA fragment among millions of other.

A solution containing DNA molecules that have the fragment of interest are introduced to a restriction endonuclease that cuts the molecules into fragments of different length.

The restriction fragments can be separated according to size by gel electrophoresis. In certain gels, fragments differing in length by just one nucleotide of several hundred can be visualised.

The DNA is then denatured by soaking the gel into alkali. The purpose is creating single stranded DNA fragments that later can be hybridized with a probe.

The gel with the single stranded DNAs is placed on top of absorbent paper. The gel is then covered with a nitrocellulose filter, and several layers of absorbent paper are placed on top of the filter.

The absorbent paper under the gel is kept wet with a concentrated salt solution that, through capillary action, is pulled up through the gel, through the filter and into the several layers of absorbent paper lying on top of everything. The concentrated salt solution is used to 'drag' the DNA up as it goes into the upper absorbent paper.  

In this process, the single-stranded DNA molecules are pulled up and bound in the nitrocellulose filter, where they have the same position as they did in the gel. They are then presented with a fx. nick-translation labelled radioactive single-stranded DNA-probe, which hybridizes with the restriction fragment that has the complementary sequence – that is our target DNA fragment.

The now radioactive DNA duplex can be visualised with autoradiography.

Restriction Fragment Length Polymorphism (RFLP)
Connor, s.25, fig. 3.2

Polymorphism – DNA point variations creating genetic diversity in the population. Site polymorphisms occur every 200-500-1000 bp in the human genome and the majority is of no clinical significance as they occur in non-coding DNA or do not result in amino acid substitutions within coding DNA, fx. in silent mutations – a polymorphism in the third base pair of a degenerated code. 

Using restriction endonuclease, ca. one in six polymorphisms can be identified.

Restriction Fragment Length Polymorphism – polymorphism that is located within the cleaving site of a restriction endonuclease. Two situations should be considered.

• If the polymorphism changes the sequence of an existing cleaving site, the restriction enzyme will not cleave and a longer restriction fragment is obtained.

• If the polymorphism creates a new cleaving site, two new shorter restriction fragments are obtained.

Shorter and longer restriction fragments change the position of the radioactive bonds obtained by southern blotting. Therefore southern blotting and the RFLP can be used in tracking inheritance of mutant and normal genes with a family. The knowledge that some of these polymorphisms lie close to specific genes and therefore are inherited together, makes RFLPs suitable as markers for specific human genome diseases.

RFLP markers can be:

• Intragenic RFLP - the restriction fragment length polymorphism can be located within the gene of interest. The polymorphism may be due to the point mutation itself or just a random polymorphism found in the intron sequence, not necesserily detecting the mutation itself.

• Extragenic RFLP – the polymorphism is located outside the gene of interest. Has a lower reliability which depends on the distance between the marker RFLP and the mutant gene, due to the possibility of mutation

Single-strand conformational polymorphism – detects modifications in the DNA molecule that are not necessarily located within a restriction endonuclease site.

Based on the fact that a single stranded DNA that is less then 400 bp long and submitted to electrophoresis through a polyacrylamide gel migrates with mobility partially dependant on its confirmation. A single base alteration usually modifies the DNA confirmation sufficiently to be detected as a mobility shift in electrophoreses. In order to compare, one needs to have a copy of the non-mutated strand.

tilbage til molekylær biologi