2. HRM REAL TIME PCR Presented by: Dadkhah Fahimeh SNP genotyping by HRM REAL TIME PCR

3. The Genomic DNA was extracted from the peripheral whole blood using a Prime Prep TM Genomic DNA Isolation Kit (Genet Bio, Korea) according to the manufacturer's instructions. Extracted DNA was used as template for amplifying the OPN and ITGA4 genes through polymerase chain reaction Preparation of primers In order to amplify OPN and ITGA4 genes, primers were designed by Primer3 software and were sent for synthesis F:5'-AAAACCAGAGGGGGAAGTGT-3' R:5'-CCCAGTAGCAAACTGAAGCTG-3' F:5'-TGCCCACTATATGCCAAAAA-3' R:5'-GGAAACTCAGAGTGGCCAGA-3 ' ' OPN ITGA4 DNA extraction

4. PCR method The PCR master mix was prepared in final volume of 25μl for n+1 samples (including negative control).

5. The micro tubes were placed in thermocycler and polymerase chain reaction was performed in 30 cycles after preparing PCR master mix

6. Gel electrophoresis method: For making 1.5% agarose gel, 0.3g of agarose powder was dissolved in 20 ml of 1x TBE buffer. The solution was heated and then the DNA green viewer dye was added to the gel. After preparing agarose gel, 6μl PCR product with 1μl loading buffer and 50bp DNA ladder were loaded to the gel. The gel was electrophoresed and then was evaluated in Gel Doc system

7. The results of electrophoresis showed a 248bp band as OPN gene PCR product and a 236bp band as ITGA4 gene PCR product.

8.  It is massively cost effective vs. other genotyping technologies such as sequencing and Taqman SNP typing.  It is fast and powerful thus able to accurately genotype huge numbers of samples in rapid time.  It is simple.  Monitoring of the DNA amplification during PCR. Advantages: Applications: SNP genotyping Mutation discovery (gene scanning) Heterozygosity screening DNA methylation analysis DNA mapping Species identification Viral/bacterial population diversity investigation HLA compatibility typing High Resolution Melting (HRM) High Resolution Melting (HRM) analysis is a new, post-PCR analysis method used for identifying genetic variation in nucleic acid sequences.