A Simple, Sensitive and Safe Method to Determine the Human α/β-Tryptase Genotype

Authors

Quang Trong Le, Virginia Commonwealth University
Sahar Lotfi-Emran, Virginia Commonwealth University
Hae-Ki Min, Virginia Commonwealth University
Lawrence B. Schwartz, Virginia Commonwealth UniversityFollow

Document Type

Article

Original Publication Date

2014

Journal/Book/Conference Title

PLOS ONE

Volume

9

Issue

12

DOI of Original Publication

10. 1371/journal.pone.0114944

Comments

Originally published at http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114944.

Date of Submission

December 2016

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.

Rights

Copyright 2014 Le et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Is Part Of

VCU Internal Medicine Publications