A simple, sensitive and safe method to determine the human ?/?-tryptase genotype.

ABSTRACT: The human tryptase locus on chromosome 16 contains one gene encoding only ?-tryptase and another encoding either ?-tryptase or the homologous ?-tryptase, providing ?:? gene ratios of 0:4, 1:3 or 2:2 in the diploid genome, these genotypes being of potential clinical relevance in severe atopy. Using an EcoRV restriction site in ?- but not ?-tryptase, PCR products, spanning intron 1 to exon 5, were used to determine ?/?-tryptase gene ratios using non-radioactive labels, including ethidium bromide labeling of all PCR products, and either digoxigenin-primer or DY682-primer labeling of only the final PCR cycle products. Sensitivity increased ?60-fold with each final PCR cycle labeling technique. Ethidium bromide labeling underestimated amounts of ?-tryptase, presumably because heteroduplexes of ?/?-tryptase amplimers, formed during annealing, were EcoRV resistant. In contrast, both final PCR cycle labeling techniques precisely quantified these gene ratios, because only homoduplexes were labeled. Using the DY682-primer was most efficient, because PCR/EcoRV products could be analyzed directly in the gel; while digoxigenin-labeled products required transfer to a nitrocellulose membrane followed by immunoblotting. This technique for determining the ?/?-tryptase genotype is sensitive, accurate, simple and safe, and should permit high-throughput screening to detect potential phenotype-genotype relations for ?/?-tryptases, and for other closely related alleles.

SUBMITTER: Le QT 

PROVIDER: S-EPMC4278853 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature