A Simple, Sensitive and Safe Method to Determine the Human alpha/beta-Tryptase Genotype

作者：Quang Trong Le; Lotfi Emran Sahar; Min Hae Ki; Schwartz Lawrence B^*

来源：PLos One, 2014, 9(12): e114944.

DOI：10.1371/journal.pone.0114944

The human tryptase locus on chromosome 16 contains one gene encoding only beta-tryptase and another encoding either b-tryptase or the homologous a-tryptase, providing alpha:beta 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 alpha-but not beta-tryptase, PCR products, spanning intron 1 to exon 5, were used to determine alpha/beta-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 similar to 60-fold with each final PCR cycle labeling technique. Ethidium bromide labeling underestimated amounts of alpha-tryptase, presumably because heteroduplexes of alpha/beta-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 alpha/beta-tryptase genotype is sensitive, accurate, simple and safe, and should permit high-throughput screening to detect potential phenotype-genotype relations for alpha/beta-tryptases, and for other closely related alleles.

• 出版日期2014-12-29