Dot-blot hybridization on PCR(Polymerase Chain Reaction) products with SSOs(Sequence Specific Oligonucleotides) have been widely used for HLA class Ⅱgenotyping. Although SSOs could discriminate one base pair difference between two alleles, major
problems with this method is that it requires a large number of specific probes and many separate hybridization procedures. It is not practical in the case of HLA typing because the number of alleles is too high. For the more convenient
genotyping,
various reverse dot-blot hybridization methods have been reported. But the direct binding of oligonucleotides to the membrane is generally not efficient, some modifications have been previously presented:poly dT-tailing, and tandemly ligated SSO
polymer.
We carried out two new technical trials to improve the reverse dot-blot hybridization. First, we synthesized olignucleotides(37bp) which contained both sequences(U-19mer primer) specific for polycloning site of pT7 Blue plasmid at the 3' end as a
primer
and SSOs(18bp) at the 5' end as a probe, This "probe-primer" and T7 promoter primer were used for PCR amplification using the pT7 Blue plasmid as a target and then the PCR products(158bp) were denatured and directly dotted on nylon membrane. This
immobilized SSOs binded efficiently to the membrane, showed no cross hybridization with HLA classⅡsequences, and were stable at room temperature for more than six months. Second, the sandwich method was used for the nonradioactive detection.
Digoxigenin labeled common probe was mixed with the PCR products which were denatured by heat before hybridization, and used for nonradioactive detection of reverse dot-blot hybridization.
This reverse dot-blot hybridization were applied to generic HLA-DQB1 genotyping, and successfully tested on 5 Homozygous Typing Cells from the Histocompatibility Workshop. Using 6 immobilized SSOs for DQB1, the following generic specificities
could
be
defined:DQB1*02, 03, 04, 05 and 06. This reverse dot-blot hybridization was used to generic DQB1 genotyping on 107 normal Koreans. The distribution of DQB1 phenotype was found as 17.8% in 0.2, 47.7% in 03, 15.9% in 04, 33.6% in 05 and 55.1% in
06.
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