Detection of 96 HLA-A, HLA-B and HLA-DR
Currently, our knowledge of the HLA system has progressed dramatically so that the characterization of HLA by genetic analysis is possible. Molecular methods such as Polymerase Chain Reaction (PCR) are now used to identify MHCs. In the PCR method, specific oligonucleotide (primers) fragments of exon sequences encoding the polymorphic regions of Class I or II MHC molecules are used to amplify and then analyze these regions.
In the HLA Typing method, several experiments are performed simultaneously on a sample using SSP-PCR. Each of these experiments examines the presence or absence of a polymorphism using specific primers for the sequence for each allele (SSP). By using the PCR technique, several versions of target and specific DNA regions in chromosome 6 that contain sequences encoding HLA alleles are produced. The system relies on the lack of exonucleic activity 5 to the 3 Taq polymerase DNA enzymes used in the PCR reaction.
To perform HLA typing using the SSP method, is essential to perform multiple reactions simultaneously. For example, there are at least 96 reactions to determine HLA-A, B, DRB1 alleles. There are also controls to prevent reproductive error and false negative results, which primers reproduce in each reaction highly protected genes (often human growth hormone).
HLA typing
There is no reproduction if there is no complementary sequence for the used primer and there is no bandage when examining the gel. Finally, the results are interpreted by comparing the reaction forms manually or using computer software designed specifically for each test.
This method is the fastest system for obtaining full HLA typing using molecular techniques that are widely available at any moment. It takes about 3.5 hours to accept the sample until the image of the gel is interpreted by DNA-based methods. In the future, this may be replaced by Real Time PCR methods that require the analysis of products using the gel. But PCR-SSP is still the fastest method based on DNA, in most HLA typing centers.