Single Base Extension

Under single base extension ( SBE), a biochemical reaction is understood that serves the genotyping of SNPs ( single nucleotide polymorphisms ). SBE is preceded by a PCR ( polymerase chain reaction ) normally.

Functional principle of the SBE

After completion of PCR and digestion is amplified ( amplified ) DNA from the regions of interest of the genome. Distinguishing features between the genomes of different people are called polymorphisms. Polymorphisms, of which only a single base is concerned, single -base mutations or SNPs may be mentioned. Different bases may lead to different gene products or altered gene expression. This can be a risk or Protektivfaktor, especially for complex diseases represent. SBE is used to determine which base is present at each of the examined person. With SBE so can determine the genotype. To this end, the digested PCR products are treated with a special SBE primers. The overall reaction of the SBE is designed so that the SBE primer only to a single base ( complementary to the SNP of interest ) is extended. This is done by adding the reaction mixture ddNTPs ( dideoxynucleoside triphosphates ) instead of dNTPs, as in PCR. At a ddNTP can be attached by polymerase due to its biochemical structure no further base. The elongation phase, which is analogous to the PCR, is thus limited to a single nucleotide. By means of subsequent measurement in the mass spectrometer, it is possible to determine to which base the SBE primer was extended because the four bases differ in their mass. The corresponding complementary base then corresponds to the desired SNP type. Also in the SBE this comes multiplexing, that is, the simultaneous investigation of different SNPs are used. Each SNP -specific SBE primer is used.

Structure of the SBE primer

SBE primer consisting of a biotin moiety, the SBE primer sequence and a photo - cleavage site. The primer sequence is designed so that it attaches itself to a specific region of DNA and ends exactly one base before the SNP. Photo- cleavage site is an area of particularly sensitive to UV light.

Implementation of the SBE

The beginning of the SBE is analogous to the PCR: It is produced a reaction mixture. The mixture consists of the SBE primer ddNTPs, magnesium chloride, and a polymerase of double-distilled water. This mixture is added to the digested PCR product. The actual reaction takes place in a thermal cycler. In thermocycler single base extension takes place. At the end of this step are in the reaction mixture of a base extended SBE primer, the unmodified PCR product remaining ddNTPs, and the other constituents of the original mixture before. With the exception of the extended SBE primer provides all of this for the subsequent measurement in the mass spectrometer represents a disturbing contamination Accordingly, followed by a purification.

Purification of the SBE products

For this purpose, the reaction mixture is added to the wells of a streptavidin plate where the biotin moiety of the SBE primer forms a strong non-covalent bond with the streptavidin and remains adhering thereto. The remaining components of the reaction mixture are removed by means of different washing buffer. In the last step there are only high-purity water and adhering to the streptavidin SBE primers in the cavity, which is then irradiated with UV light. Thereby breaking the SBE primer on the photo- cleavage site. The mass of a base extended primer - breaking piece can now be determined in a mass spectrometer.

• Molecular Biology
• Biological test method