Methodology flow diagram. In step 1, unique probes competitively hybridize to genomic DNA, designed with a tail including unique spacer sequences (labeled X), and common PCR primer sequences. Taq DNA ligase is used in step 2 to join probe pairs that had hybridized adjacent to one another. Only successfully joined probe pairs were amplified by PCR in step 3 using the common PCR primer sequences. Extended PCR product oligonucleotides are represented by a broken line. In step 4, all PCR-amplified probe pairs were separated and visualized using capillary fragment analysis. The presence or absence of each probe pair ligated determines the HLA-DQB1 allelotypes as noted in table 1. This example data with probe pairs 1, 2, 5, 8, 9, and both controls amplified was assigned the allelotype DQB1*02/*06D.

Example of allelotyping data. Peaks derived from ligated and amplified probe pairs can be clearly distinguished from the background (BLANK). The small peaks at ~18 nucleotides are the unincorporated fluorescently labeled left PCR primer. C1 and C2 are positive DNA control probes. Size in nucleotides is normalized based on an added ROX dye labeled DNA ladder (Standard ladder). Peak output data from probe mix A and probe mix B is combined to determine allelotypes. Here, we’ve chosen to show examples of data where just one probe mix contained all relevant probes, for simplicity of presentation.