%0 Journal Article
%T Real-time PCR detection of PI*S and PI*Z alleles of SERPINA1 gene using SYBR green.
%A Ramos-Díaz R
%A Escuela-Escobar A
%A Díaz-Usera A
%A Hernández Pérez JM
%A González-Carracedo MA
%A Pérez-Pérez JA
%J Gene
%V 921
%N 0
%D 2024 Aug 30
%M 38723785
%F 3.913
%R 10.1016/j.gene.2024.148540
%X <B>BACKGROUND: </B>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.<BR><B>OBJECTIVE: </B>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.<BR><B>METHODS: </B>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.<BR><B>RESULTS: </B>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.<BR><B>CONCLUSIONS: </B>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.