High Resolution Melt Curve Analysis for the detection of A1, A2 β-casein variants in Indian Cows (original) (raw)

Genetic variants in bovine β-casein gene (A1 and B) at codon 67, encode a histidine residue resulting in potential release of a bioactive peptide, β-casomorphin-7 upon digestion, responsible for many human disorders like Type 1 diabetes, autism and heart diseases. Present study includes development of High Resolution Melt Curve Analysis to detect single nucleotide substitution characterizing the A1/A2 alleles of the CSN2 gene. 314 hair follicles were considered for the development of High Resolution Melt curve analysis for differentiating A1, A2 beta casein alleles.100 base pairs amplicon in 301 DNA hair follicles confirmed the presence of β-casein gene. Sequencing of eighty amplicons were done which showed that 24 were A2 homozygous (A2A2), 11were A1 homozygous (A1A1) and 37 heterozygous (A1A2). The sequenced data was further utilized for the development of difference plots and melt curves with HRMA utilizing Real Time PCR. All the sequenced samples were subjected for HRMA where 11(13%) were A1A1 homozygous, 21(27%) A2A2 homozygous and 37 (60%) A1A2 heterozygous. The HRMA results were also in agreement with similar results confirmed by sequencing. The technique is a fast, accurate, novel and inexpensive genotypic identification assay which can analyze large number of specimens without the requirement for post-amplification processes.