Real-time PCR detection of PI*S and PI*Z alleles of SERPINA1 gene using SYBR green
Ruth Ramos-Díaz1, Ainhoa Escuela-Escobar2, Ana Díaz-Usera3
1Fundación Canaria Instituto de Investigación Sanitaria de Canarias (FIISC) - Unidad de Investigación Complejo Hospitalario Universitario de Canarias, Servicio de Farmacia, Tenerife, Spain.
Background:
Alpha-1 antitrypsin deficiency is an underdiagnosed genetic condition that predisposes to pulmonary complications and is mainly caused by rs28929474 (PI*Z allele) and rs17580 (PI*S allele) mutations in the SERPINA1 gene.
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.
