Differentiating between monozygotic twins through methylation specific high resolution melt curve analysis

Abstract

Whilst DNA profiling is an extremely powerful human identification tool, it is limited when it comes to monozygotic (MZ) or identical twins, where in such cases they would exhibit the same DNA profile. This creates issues where the suspect is one of the twins. Currently, efforts are being made to resolve this through whole genome or next generation sequencing, which can be very expensive. This study explores the feasibility of using high resolution melt curve analysis to characterize the DNA methylation status. DNA methylation can be altered by environmental factors, consequently over the life of monozygotic twins; they can end up with differing DNA methylation patterns, typically characterized by sequencing. Buccal swabs were collected from five sets of MZ twins of differing ages, as well as from a full sibling (non-MZ). All samples underwent DNA extraction, bi-sulfite treatment, amplification and then underwent high resolution melt curve analysis using a 7500 Fast Real-Time PCR machine, targeting a section of the Alu-SP region and the promoter region of the E2F3 gene. Significantly different melting temperatures (Tm) were obtained in four of the five MZ twins of the alu- SP samples, as well as in the non-MZ full sibling samples, and all 5 twin sets of samples analysing the E2F3 fragment; thus indicating that a relatively cheaper faster test could be used to differentiate between MZ twins in a forensic context