Dot-blot hybridiszation on PCR(Polymerase Chain Reaction) products with SSOs(Sequence-Specific Oligonucleotides) have been widely used for HLA class II genotyping. 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 oligonucleotides(37bp) which contained both sequences(U-19mer primer) specific for polycloning site of pT7blue plasmid(Novagen Co.) 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(158 bp) were denatured and directly dotted on
nylon
membrane. This immobilized SSOs binded efficiently to the membrane, showed no cross hybridization with HLA class II sequences, and were stable at room temperature for more than six months. Second, the HLA-DQA1 gene was amplified in the presence
of
digoxigenin-dUTP by PCR. Digoxigenin labelled PCR products wee denatured by head before hybridization and used for nonradioactive detection of reverse dot-blot hybridization.
This reverse dot-blot hybridization were applied to generic HLA-DQA1 genotyping, and succesfully tested on 7 Homozygous typing Cells from 10th Histocompatibility Workshop. Using 8 immobilized SSOs for DQA1, the following generic specificities
could
be
defined:DQA1*01, 01, 03, 04, 05, and 06. This reverse dot-blot hybridization was used to generic DQA1 genotyping on 93 normal Koreans. These samples wee simultaneously genotyped using two-step PCR method previously presented by our laboratory.
The
results, by both of the two methods. Were completely matched.
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