DNA probes for HLA typing

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roudi's picture
DNA probes for HLA typing

DNA probe arrays and primers for HLA class I and II typing have been around for a while. I think they work based on the reverse hybridization principle? Can someone explain this to me and if possible some published articles regarding reverse hybridization ?

RH's picture
Typer's picture
In a nutshell...

In a nutshell...

Classic hybridization involves immobilizing a target sequence on solid support (nylon or nitrocellulose membrane is what we used back in the day..), then probing with a specific probe sequence after denaturing the immobilized target. Think classic Southern blotting for large restriction enzyme cuts of genomic, bacterial, viral DNA.

In reverse hybridization the probe sequence is immobilized and the target arrives in solution. Often these probes are now oligonucleotides and the targets are PCR products. Thus for HLA typing, the PCR products are generated by primers flanking a large area of HLA gene sequence, and the probe oligos are designed to discriminate the polymorphisms between the primers. You determine which variations are present in the sample by which probes light up.

The advantage of reverse-hyb over the competing method (SSP PCR) is that for each donor or recipient a relatively small number of PCR reactions can be used to generate the typing, thus allowing higher throughput. This type of assay can be done on nitrocelluose, on microarrays, or on styrene beads - those are just the commercial kits for HLA typing that I've seen.

Hope this helps....