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The Past, Present and Future of HLA Typing

e HLA DNA directly through differential primer extension.

One of the most commonly employed strategies in HLA DNA typing is to first amplify a given HLA locus, then hybridize it with a labeled, sequence specific oligonucleotide probe. The original methods, described in the 1980s, first blotted PCR amplified DNA onto nitrocellulose or nylon filters. These dot blots could then be incubated with radioactively labeled, sequence specific oligonucleotide probes. These probes would bind only to complementary HLA DNA amplified by PCR. Under appropriate conditions, sensitivity and specificity could be high enough to detect single nucleotide differences consistently. Several probes could be used to identify an array of HLA alleles. Today, radioactive probes are seldom used, and sequence specific oligonucleotides are now labeled with fluorescent dyes, biotin, digoxigenin, or directly with enzymes such as horseradish peroxidase. The appropriate substrate can be added to produce a fluorometric (or colorimetric) readout without the problems associated with radioactivity. Variations of the technique have also risen; for example, it is possible to construct PCR primers pre labeled with biotin, and then amplify biotin labeled HLA loci. These PCR products are then incubated with unlabeled, sequence specific oligonucleotide probes immobilized on the membrane. After the incubation and wash steps, the hybridization products can be detected by streptavidin horseradish peroxidase and chromogenic substrate. Modifications such as this allow the HLA typing to be completed in two steps with high accuracy.

The second HLA DNA typing technique is to use the PCR amplification reaction directly to detect HLA polymorphisms. In this technique, primers can be constructed specifically to complement HLA polymorphisms; if the primers bind the complementary polymorphism and amplify the gene segment, then the PCR product can be detected by standard techniques. If the p
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