HRM for genotyping
Genotyping in animal, plant, microbial species, strain screening, screening, defects, detection of mutant genes, accurate drug use such as cancer treatment, monitoring, etc., and consumer gene detection such as alcohol metabolism according to individual gene types There are many applications in areas such as capacity gene detection.
Typically, genotyping SNPs employ a fluorescent probe endpoint method. Two fluorescent probes are required to detect one site, such as a fluorescent reporter group for one probe using a FAM channel and the other with a HEX (VIC) channel. Due to the high cost of synthesizing PCR fluorescent probes, in order to reduce costs, a smaller reaction system can be used, such as a 5 uL reaction system on a 384-well real-time PCR instrument. For some non-clinical and consumer, or cost-sensitive genotyping applications that need to detect more than tens of sites, consider using a high-resolution melting curve method for saturated dyes (HRM). 0.1 ° C), if the cost of nucleic acid extraction is not included, the cost can be reduced by 10 times or even 100 times compared with the probe method. In order to distinguish two DNA fragments which differ by only one base at the melting temperature Tm, the HRM method selects the length of the amplified DNA fragment as short as possible, so that, in addition to the design of the primer, In addition to the special requirements of short amplified fragments, there is a high requirement for the HRM performance and accurate performance of the real-time PCR instrument. The following points are key to ensuring that HRM achieves correct and reliable genotyping.
First of all, the resolution of HRM is better than 0.05 °C. Generally, only the QPCR instrument with fluorescence imaging technology can only perform the signal of all sample wells at the same time, and the scanning of the instrument requires a total of 5 More than two seconds, that is, the detection time difference between the first hole and the last hole is more than 5 seconds, and the temperature is continuously changed during the melting process, which will result in inconsistent temperature detection on each hole every time, therefore, scanning type QPCR instruments are generally not suitable for HRM.
Secondly, in order to use primers that only generate amplification products smaller than 100 bp, sometimes the specificity may not be well balanced, and the specificity of the HRM dye method itself is weak, which brings the non-specificity to the fluorescence quantitative PCR experiment. In addition to the need to select a QPCR Master Mix with good amplification specificity, it is also necessary to use a gradient QPCR pre-experiment to optimize the annealing temperature, find the optimal annealing temperature of the primer, and then use Touchdown PCR or TD PCR to effectively Control non-specific amplification. Stepdown PCR also inhibited non-specific amplification, but the inhibitory effect was weaker than Touchdown PCR.
Third, if it is necessary to distinguish the difference between the Tm of two homozygotes, for example, when the Tm difference between two homozygotes is only 0.5 °C, their corresponding heterozygotes may be difficult to distinguish between two homozygotes by simply relying on Tm. At this time, we can introduce the Tm peak width factor of the melting curve or the line type of the peak to judge the heterozygote.
Figure 1 shows the SNP typing results of 384 samples at one site using the endpoint method of FAM and VIC probes on a FS384 QPCR instrument from Fusheng Biotechnology using a 5 uL reaction system.
Figure 2 shows the low-cost genotyping using the Touchdown PCR method on Fusheng Bio's FS384 QPCR instrument, followed by high-resolution 0.02 °C HRM. The table at the bottom left of the figure lists all sample wells. The typing results of each sample were correctly determined based on the Tm value and its peak width value. In this experiment, the peak widths of both homozygotes were less than 1.5 ° C, while the peak width of heterozygotes was around 2 ° C.
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