Single base extension
Under single-base extension ( SBE ) a biochemical reaction is understood that the genotyping of SNPs ( single nucleotide polymorphisms is used). The SBE is usually preceded by a PCR ( polymerase chain reaction ).
Functional principle of the SBE
After performing the PCR and digestion , amplified (replicated) DNA from the regions of the genome of interest is available. Polymorphisms are the distinguishing features between the genomes of different people. Polymorphisms that only affect a single base are called single base mutations or SNPs. Different bases can lead to different gene products or altered gene expression . This can represent a risk or protective factor, especially for complex diseases. The SBE is used to determine which base is present in the person being examined. The genotype can therefore be determined using SBE. For this purpose, the digested PCR products are mixed with a special SBE primer . The overall reaction of the SBE is designed in such a way that the SBE primer is only extended by a single base (which is complementary to the SNP of interest). This is done by adding ddNTPs (dideoxy nucleoside triphosphates ) to the reaction mixture instead of dNTPs as in the PCR. Due to its biochemical structure, no further base can be attached to a ddNTP by polymerase . The elongation phase, which takes place analogously to the PCR, is therefore limited to a single nucleotide. By means of a subsequent measurement in the mass spectrometer , it is possible to determine by which base the SBE primer was extended, since the four bases differ in their mass. The corresponding complementary base then corresponds to the sought-after SNP type. The SBE also uses multiplexing, i.e. H. the simultaneous examination of different SNPs. A specific SBE primer is used for each SNP.
Structure of the SBE primer
The SBE primer consists of a biotin residue, the SBE primer sequence and a photo cleavage site. The primer sequence is constructed in such a way that it attaches to a specific region of the DNA and ends exactly one base before the SNP. The photo cleavage point is an area that is particularly sensitive to UV light.
Implementation of the SBE
The start of the SBE is analogous to the PCR: a reaction mixture is prepared. The mixture consists of the SBE primer, ddNTPs, magnesium chloride, a polymerase and double distilled water. This mixture is added to the digested PCR product. The actual reaction takes place in a thermal cycler . The single base extension takes place in the thermal cycler. At the end of this step, the reaction mixture contains the SBE primer extended by one base, the unchanged PCR product, remaining ddNTPs and the other components of the original mixture. With the exception of the extended SBE primer, all of this represents a disruptive contamination for the subsequent measurement in the mass spectrometer. A purification follows accordingly .
Purification of the SBE products
For this purpose, the reaction mixture is poured into the cavities of a streptavidin plate, where the biotin residue of the SBE primer forms a strong non-covalent bond with the streptavidin and adheres to it. The remaining components of the reaction mixture are removed using various washing buffers. In the last step, only ultrapure water and the SBE primer adhering to the streptavidin are in the cavity, which is now irradiated with UV light. This breaks the SBE primer at the photo cleavage site. The mass of the primer fragment lengthened by one base can now be determined in the mass spectrometer.
Individual evidence
- ↑ GA Denomme: Single base extension in multiplex blood group genotyping. In: Methods in molecular biology (Clifton, NJ). Volume 496, 2009, pp. 15-24, ISSN 1064-3745 . doi : 10.1007 / 978-1-59745-553-4_2 . PMID 18839101 .
- ↑ L. Kaderali: Primer design for multiplexed genotyping. In: Methods in molecular biology (Clifton, NJ). Volume 402, 2007, pp. 269-286, ISSN 1064-3745 . doi : 10.1007 / 978-1-59745-528-2_13 . PMID 17951800 .