HLA Typing with Sequence-Specific Oligonucleotide Primed PCR (PCR-SSO) and Use of the Luminex™ Technology

Abstract

The hybridization products obtained by PCR using sequence-specific oligonucleotides can be traced either by colorimetric (streptavidin–biotin)-, X-ray (digoxigenin–CSPD)-, or fluorescence (FITC, PE)-based detection systems. To achieve a faster, reliable, automated typing technique microbead and fluorescence detection technology have been combined and introduced to this field (XMAP™ technology). For each locus, a series of microspheres, which are recognizable by their specific color originating from two internal fluorescent dyes, are used. Each microsphere is coupled with a single probe that is capable of hybridizing with the biotin-labeled complementary amplicon. Once hybridization occurs, it can be quantified by measuring the fluorescence signal originating from fluorescently (streptavidin–PE) labeled amplicons captured by the beads. Currently, there are two commercially available systems that differ in the scale of probes and the methods used for amplification and denaturation. One of these is described in detail in this chapter.