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查看更多产品信息 High Resolution Melt Software v3.0.1 - FAQs (4461456)
6 个常见问题解答
We recommend to use 50-250 bp long PCR amplicons. Typically, shorter amplicons can distinguish the genotypes for a SNP better, especially for Type III and Type IV SNPs. This is simply because a single base variation affects the melting behavior of a 100 bp amplicon stronger than of a 500 bp amplicon, for example. In longer fragments, the risk of covering multiple mutations is also increased.
Positive control reactions should contain template DNA with a known sequence. In SNP genotyping experiments, this could be a sample with a known genotype. Positive control(s) for all genotypes should be included where possible to serve as a reference in melting curve comparison and assigning genotypes for test samples. In mutation scanning experiments, a sample with a wild type sequence could serve as a positive control. The controls should preferably have the same DNA concentration as their corresponding test samples. Control DNA should also be eluted and/or diluted in the same buffer as the samples.
A 3-step cycling protocol is recommended for the analysis of complicated (especially Type IV SNP) targets, amplicons longer than 200 bp, and amplicons with a primer annealing temperature that is less than 60 degrees C.
HRM can be performed on the 7500 Fast, 7900HT Fast, StepOne/StepOnePlus instruments and requires a separate purchase of HRM software. The following instruments are also compatible with HRM and have a built-in analysis module within the instrument software: ViiA7, QuantStudio 6 and 7 Pro, QuantStudio 6 and 7 Flex, QuantStudio 12K Flex, QuantStudio 1, QuantStudio 3, and QuantStudio 5. To activate the HRM module, you will need to purchase a license.
To perform an HRM experiment, you will need the reagents to calibrate your instrument for the HRM dye and the software to perform the analysis.
High resolution melt (HRM) can be used in genotyping, mutation scanning, and methylation studies.