title: A simple, sensitive and safe method to determine the human α/β-tryptase genotype.
abstract: The human tryptase locus on chromosome 16 contains one gene encoding only β-tryptase and another encoding either β-tryptase or the homologous α-tryptase, providing α:β 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 α- but not β-tryptase, PCR products, spanning intron 1 to exon 5, were used to determine α/β-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 α-tryptase, presumably because heteroduplexes of α/β-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 α/β-tryptase genotype is sensitive, accurate, simple and safe, and should permit high-throughput screening to detect potential phenotype-genotype relations for α/β-tryptases, and for other closely related alleles.
authors.affiliation: Department of Internal Medicine, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Quang Trong
authors.initials: QT
authors.lastname: Le
authors.affiliation: Department of Internal Medicine, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Sahar
authors.initials: S
authors.lastname: Lotfi-Emran
authors.affiliation: Department of Internal Medicine, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Hae-Ki
authors.initials: HK
authors.lastname: Min
authors.affiliation: Department of Internal Medicine, Virginia Commonwealth University, Richmond, Virginia, United States of America.
authors.forename: Lawrence B
authors.initials: LB
authors.lastname: Schwartz
mesh_terms: D000328:Adult; D002885:Chromosomes, Human, Pair 16; D005796:Genes; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D053802:Tryptases / Q000235:genetics*; D055815:Young Adult
chemical_list: D053802:Tryptases