Application of silver staining to the rapid typing of the polymorphism of HLA-DQ alleles, by enzymatic amplification and allele-specific restriction fragment length polymorphism
✍ Scribed by Dr. Li-Ya Ju; Xue-Fan Gu; Etienne Larger; Rajagopal Krishnamoorthy; Dominique Charron
- Publisher
- John Wiley and Sons
- Year
- 1991
- Tongue
- English
- Weight
- 479 KB
- Volume
- 12
- Category
- Article
- ISSN
- 0173-0835
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✦ Synopsis
Application of silver staining to the rapid typing of the polymorphism of HLA-DQ alleles by enzymatic amplification and allele-specific restriction fragment length polymorphism
A rapid and highly sensitive silver staining method, originally developedfor the detection of proteins, was slightly modified to detect nucleic acids in polyacrylamide gels. The second exons of the histocompatibility antigen HLA-DQA 1 and DQB 1 genes were selectively amplified from genomic DNA by the polymerase chain reaction (PCR). Digestion of the PCR products by endonucleases, followed by their sizeseparation on polyacrylamide gels and visualization by silver staining, allowed us to define the HLA-DQ alleles of the genomic DNA. The intensity of staining of digested PCR-amplified D N A is linear from at least 8 to 18 ng for fragments oflengths ranging from approximately 40 to 200 bp. Thus, silver staining in combination with PCR and allele-specific restriction fragment length polymorphism provides a simple, safe, and rapid method for accurate definition of HLA-DQ alleles at the nucleotide level in the clinical typing laboratory.