High-resolution melting curve (HRM) analysis of premixed Mix (PCR), can genetic screening?

       High-resolution melting (HRM) is a new gene analysis technique based on the melting temperature of single nucleotides to form different morphological melting curves. It has extremely high sensitivity and can detect single bases. difference.

 

       As an efficient and robust "post-PCR" technology, it is not limited by the position and type of mutated bases. Without the need for sequence-specific probes, high-resolution melting can be run directly after PCR to complete the analysis of the sample. HRM technology has received widespread attention due to its ease of operation, low cost, accurate results, and true closed-tube operation.

       HRM principle:

       A novel DNA-saturated fluorescent dye is added to the reaction system before the PCR reaction, and then the PCR product is directly introduced into a high-resolution melting analyzer, and the PCR amplification product is subjected to heat denaturation in a certain temperature range, and the optical detection system of the instrument is collected and analyzed. The fluorescence signal is plotted and the temperature melting curve is plotted to distinguish (single) base differences based on the shape of the different melting curves.

 

The most widely used application area for HRM analysis is genetic screening. Genetic screening is the search for the presence or absence of an unknown mutation in a PCR amplified fragment, either before the sequencing step or as an alternative to sequencing. Since mutations in the PCR product can cause a change in the shape of the DNA melting curve, it can be detected using HRM analysis. When a hybrid double-stranded DNA sample is amplified and melted, its melting curve is different from that of a homozygous wild-type or mutant sample. If done properly, you can reduce your sequencing effort by at least 95%. With the development of DNA dyes and analytical instruments, HRM technology will continue to be improved and improved, playing a more important role in the molecular diagnostic field of genetic diseases.

 

       HRM application:

 

High Resolution Melting Curve Analysis Technology (HRM) Questions and Answers

 

As a high quality reagent supplier specializing in the field of PCR for more than 24 years, Solis BioDyne recommends it, 5X hot start EvaGreen high resolution melting curve analysis - premixed Mix (PCR):

       * Does not contain reference dye ROX, if you need ROX, please select: 08-33-00001

 

       5X Hot Start EvaGreen High Resolution Melting Curve Analysis - Premixed Mix (PCR) provides the following components:

 

HOT FIREPol® DNA Polymerase 5x EvaGreen®HRM buffer 12.5 mM MgCl2 = 1x PCR solution – 2.5 mM MgCl2 dNTPs EvaGreen®dye BSA (Customers only need to prepare water, template DNA, primers)

       Very high cost performance (import quality, domestic price):

 

       Recommended qPCR reaction system configuration:

       * DNA template concentration: cDNA 0.1 pg/μl -10 ng/μl; gDNA 10 pg/μl – 4 ng/μl

 

       Recommended qPCR reaction procedure:

 

       Published article experimental results show:

       [1] Negrisolo, Susanna et al. “Could the interaction between LMX1B and PAX2 influence the severity of renal symptoms?” European Journal of Human Genetics 26 (2018): 1708-1712.

Analysis of missense mutations in exon 4 of PAX2 gene using Solis kit

         [2] Gaczkowska, Agnieszka, et al. “Association of CDKAL1 Nucleotide Variants with the Risk of Non-Syndromic Cleft Lip with or without Cleft Palate.” Journal of Human Genetics, vol. 63, no. 4, 2018, pp. 397 –406.

Analysis of CDKAL1 gene SNP using Solis kit

         The key to the success of HRM analysis:

       1. Maintain a shorter amplified fragment for maximum sensitivity. Compared to larger amplified fragments, amplified fragments of about 100 bp can simplify the detection of single nucleotide melting curves.

       2. Ensure the specificity of the PCR amplified fragment. Mismatched products and primer dimers can complicate data interpretation. Primer concentrations below 200 nM, MgCl2 in the 1.5 mM to 3 mM range and the use of hot-start DNA polymerase will help achieve high specificity.

       3. Make sure the reaction uses enough templates. In general, Ct should be less than 30 to generate enough material for accurate melt analysis.

       In addition to the highly efficient and robust HRM analysis premixed Mix products described above, Solis BioDyne provides customers with end-point PCR, qPCR (real-time PCR), reverse transcription (Reverse transcription), and other PCR Supporting reagents. The product line overview is as follows:

Solis BioDyne is from the European Union-Estonia and has been specializing in the field of PCR for more than 24 years. Solis BioDyne has been developing and producing high quality life science reagents and is now one of the leading reagent suppliers in Europe today. High standards of production standards and services make Solis BioDyne the trusted PCR supplier of choice worldwide. Our DNA polymerases, PCR Master Mixes, qPCR Mixes and other reagents are used by researchers in more than 110 countries around the world, including leading research institutes and biotechnology companies.

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