Le Quang Trong, Lotfi-Emran Sahar, Min Hae-Ki, Schwartz Lawrence B
Department of Internal Medicine, Virginia Commonwealth University, Richmond, Virginia, United States of America.
PLoS One. 2014 Dec 29;9(12):e114944. doi: 10.1371/journal.pone.0114944. eCollection 2014.
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.
位于16号染色体上的人类类胰蛋白酶基因座包含一个仅编码β-类胰蛋白酶的基因，以及另一个编码β-类胰蛋白酶或同源α-类胰蛋白酶的基因，在二倍体基因组中提供的α:β基因比率为0:4、1:3或2:2，这些基因型在严重特应性疾病中具有潜在的临床相关性。利用α-类胰蛋白酶而非β-类胰蛋白酶中的EcoRV限制性酶切位点，跨越内含子1至外显子5的PCR产物被用于使用非放射性标记物来确定α/β-类胰蛋白酶基因比率，包括对所有PCR产物进行溴化乙锭标记，以及仅对最终PCR循环产物进行地高辛引物或DY682引物标记。每种最终PCR循环标记技术的灵敏度提高了约60倍。溴化乙锭标记低估了α-类胰蛋白酶的量，推测是因为退火过程中形成的α/β-类胰蛋白酶扩增子异源双链体对EcoRV具有抗性。相比之下，两种最终PCR循环标记技术都能精确地定量这些基因比率，因为只有同源双链体被标记。使用DY682引物最为有效，因为PCR/EcoRV产物可直接在凝胶中分析；而地高辛标记的产物需要转移到硝酸纤维素膜上，然后进行免疫印迹。这种确定α/β-类胰蛋白酶基因型的技术灵敏、准确、简单且安全，应该能够进行高通量筛选，以检测α/β-类胰蛋白酶以及其他密切相关等位基因的潜在表型-基因型关系。
