ISSN:
1744-313X
Quelle:
Blackwell Publishing Journal Backfiles 1879-2005
Thema:
Biologie
,
Medizin
Notizen:
We describe a new, non-radioactive microtitre plate assay for the analysis of genetic variations at the DNA level. The new method combines hybridization of oligonucleotides with PCR amplified DNA in liquid phase with detection in solid phase using an elisa-reader. Genomic DNA is labelled with digoxigenin during PCR using a nucleotide mix containing DIG-11-2′-deoxy-uridine-5′-triphosphate (DIG-11-dUTP) DIG labelled, amplified genomic DNA is hybridized in solution with an oligon de which is labelled with one biotin at its 3′-end, using biotin-16,2′,3′-dideoxy ine-5′-triphosphate (BIO-16-ddUTP) and DNA deoxynucleotidylexo-transferase (TdT). The hybridized complex is immobilized in a streptavidin (SA) coated microtitre plate via the biotin and detection of digoxigenin is performed using anti-digoxigenin horseradish peroxidase, fab fragments (anti-DIG-POD), and the colorimetric substrate 2,2′-Azino-di-(3-ethylbenzthiazolinsulfonat[6]) (ABTS®). The resulting absorbtion of the assay is analysed in a microtitre plate reader. This method results in highly specific and sensitive hybridization signals and with the 15 oligonucleotides chosen, allows the typing of DR1-DR10.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1111/j.1744-313X.1993.tb00161.x
