OBJECTIVE: Development of a homogeneous genotyping test for detection of PI*S and PI*Z alleles based on the principles of allele-specific PCR and amplicon melting analysis with a fluorescent dye.
METHODS: Sixty individuals, which included all possible genotypes that result from combinations of rs28929474 and rs17580 single nucleotide variants, were assayed with tailed allele-specific primers and SYBR Green dye in a real-time PCR machine.
RESULTS: A clear discrimination of mutant and wild-type variants was achieved in the genetic loci that define PI*S and PI*Z alleles. Specific amplicons showed a difference of 2.0 °C in melting temperature for non-S and S variants and of 2.9 °C for non-Z and Z variants.
CONCLUSIONS: The developed genotyping method is robust, fast, and easily scalable on a standard real-time PCR platform. While it overcomes the handicaps of non-homogeneous approaches, it greatly reduces genotyping costs compared with other homogeneous approaches.
目的:基于等位基因特异性PCR和荧光染料扩增子解链分析的原理,开发用于检测PI*S和PI*Z等位基因的均质基因分型试验。
方法:60个人,其中包括由rs28929474和rs17580单核苷酸变体组合产生的所有可能的基因型,在实时PCR仪中用加尾等位基因特异性引物和SYBRGreen染料进行检测。
结果:在定义PI*S和PI*Z等位基因的遗传基因座中实现了对突变体和野生型变体的明确区分。特定扩增子显示非S和S变体的解链温度差异为2.0°C,非Z和Z变体的解链温度差异为2.9°C。
结论:开发的基因分型方法是稳健的,快,并且在标准的实时PCR平台上易于扩展。虽然它克服了非同质方法的障碍,与其他同质方法相比,它大大降低了基因分型成本。