Clara HRM Mix

High resolution melting analysis of 4 SNPs using Clara HRM Mix. A. SNP rs12913832 (G/A) associated with the expression of the OCA2 gene; B. SNP rs2230199 (C/G) associated with the expression of the C3 gene; C. SNP rs641805 (A/T) associated with the expression of the DPYD gene; D. SNP rs9903378 (A/C) associated with the expression of the TP53 gene. Reactions include 0.4 µM of each primer and 5 ng of human genomic DNA. Cycling conditions were 95°C 2 min followed by 45 cycles of 95 °C 5 s and 60 °C 20 s.

Super senstitive melt curve separation allows accurate distinction of class I to class IV SNPs with Clara HRM Mix.

Using HRM qPCR for methylation analysis.
•Bisulfite (HSO3-) treated cytosine (C) residues are converted to uracil (U)
•This reduces PCR product melt points because UA/TA bp are less stable than CG bp
•Methylated PCR products retain a high melt temperature after treatment
•Unmethylated PCR products shift to lower melt temperatures after treatment.

DNA Methylation analysis of FN3K gene by high resolution melting. Human genomic DNA with different levels of methylation (0%-100%) was treated with bisulfite prior to qPCR amplification. Clara® HRM Mix was used in qPCR reactions which included 0.4 µM of each primer and 5 ng of human genomic DNA. Cycling conditions were 95°C 2 min followed by 45 cycles of 95 °C 15 s, 60 °C 20 s, and 72 °C 10 s on a Roche Applied Science Lightcycler® 96.

Clara® HRM Mix enables accurate estimation of CpG methylation in DNA targets.