Application prospect of the hottest high resolutio

2022-07-29
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Application prospect of high-resolution melting HRM

high resolution melting (HRM) analysis is a new technology. It can detect the small sequence differences of PCR fragments only by analyzing the fusion curve after PCR, so it can be used in mutation scanning, sequence pairing and genotyping. With its speed and simplicity, the method of high-resolution fusion is rapidly becoming popular

in fact, the melting analysis of double stranded DNA can be traced back to the 1960s, when it was monitored by ultraviolet absorption. The analysis requires a few micrograms of DNA, while the sample takes 0 Heating at a rate of 0 ° c/minute is slow and often takes several hours to complete. Later, the appearance of lightcycle quantitative PCR made fluorescence fusion analysis popular. The sample volume is reduced to the nanogram level due to capillary tube, and the melting rate is faster, up to 0 0 ° c/s, which reduces the melting time to a few minutes. The dye used at that time was SYBR Green I

however, this fusion analysis can only distinguish DNA sequences with significant differences in fragment size and GC content, such as checking whether there are primer dimers and other nonspecific amplification in PCR amplification products. If you want to distinguish SNPs, the resolution is not enough. Why? It is necessary to introduce saturated dyes and unsaturated dyes first. Sybrgreeni is an unsaturated dye. Due to its inhibition on PCR, the concentration used in the experiment is very low, far from saturating the small groove in the DNA double helix structure. In this way, when the DNA double strand is denatured at high temperature, the fluorescent dye molecules in the single strand part rearrange, and the fluorescent dye molecules recombine to the vacant sites of the double strand DNA, resulting in no change in the fluorescent signal, so false negatives occur and the specificity decreases

thus, saturated dyes came out. Why is it called saturated dye? Because they do not inhibit PCR even at saturation concentration (maximum fluorescence). Therefore, a high concentration of dye saturates the small grooves in the DNA double helix structure, so that no rearrangement will occur during the process of DNA unwinding, so that the melting curve has a higher resolution. At present, the saturated dyes on the market mainly include lcgreen, LC green plus, SYTO 9, etc. Dye alone is not enough. The instrument should be upgraded accordingly

the fusion curve of the amplicon depends entirely on the DNA base sequence. If one base in the sequence is mutated, the unwinding temperature of the DNA strand will be changed. But the difference is very small, only a few tenths of a degree Celsius. If the resolution of the instrument is not high, it can not be detected at all

what kind of resolution is high resolution? According to the existing function test of the instrument, at least 10 data points shall be obtained per degree Celsius during the melting operation, which is the minimum requirement for the instrument. In addition, temperature uniformity is also important. If the temperature difference between the two holes is 0.1 ° C, it is likely to lead to the final melting temperature difference of 0.1 ° C, so the accuracy of HRM analysis results cannot be guaranteed. The inter hole temperature difference of most conventional quantitative PCR instruments is 0 5 ° C, which also determines that they are not competent for HRM

at present, many manufacturers in the market provide HRM analysis instruments, including idahot and the special use environment of materials, which put forward higher requirements for the experimental conditions of material testing machines. Some are dedicated to HRM, and some are quantitative PCR instruments with HRM functions. The inventor of HRM, Wittwer Laboratory of the University of Utah School of medicine, USA, has evaluated the performance of several instruments on the market in genotyping and mutation scanning. 1. They found that for the accurate genotyping of most instruments, the main limitation is the spatial temperature difference on the plate (the standard deviation of TM is 0..264 ° C). Other differences such as data density, signal-to-noise ratio and fusion rate will also affect the scanning of heterozygotes. Through comparison, it is found that the air heated instrument and the instrument whose sample temperature is controlled separately have lower TM standard deviation (0..065), while the heated block instrument is at 0 274。

with saturated dyes and high-resolution instruments, HRM analysis can begin. This process is actually very simple, that is, the PCR amplicon is heated, and the temperature gradually rises from 50 ° C to 95 ° C. During this process, the amplicon gradually disintegrates, and when the fusion temperature (TM) is reached, the DNA strands are completely separated. At the initial stage of HRM analysis, the fluorescence intensity was very high. With the increase of temperature, the double stranded DNA gradually decreased, and the fluorescence intensity also decreased. HRM instrument records the whole process of fluorescence change through camera. By plotting the data, a melting curve is generated. The vortex flowmeter

is really simple enough to ask for more detailed technical data. There is no need to design the probe, just add a saturated dye to the conventional PCR. HRM can not only screen and scan unknown mutations, but also analyze known mutations. Compared with the traditional catastrophe analysis, the operation steps are greatly simplified, and the time and cost are also reduced. Moreover, the samples were directly analyzed by HRM after PCR amplification without transfer, which truly realized the closed tube operation and reduced the risk of pollution

with these advantages, HRM has been widely used in mutation scanning, sequence pairing, genotyping, methylation analysis and other applications in recent years

sequence pairing

common methods for sequence difference analysis include single strand conformation polymorphism, denaturing gradient gel electrophoresis, denaturing high performance liquid chromatography, sequencing, etc. these methods require separation of samples, followed by multi-step reactions, which are cumbersome and time-consuming, and the PCR products are at risk of contamination. However, HMR can be completed within 5 minutes without additional steps. It is also easy to develop into high-throughput screening. It is the only closed tube operation, which increases the reliability of analysis. These advantages are particularly important for molecular diagnostic analysis

in organ transplantation, doctors usually test the HLA of siblings to see whether the major histocompatibility matches. By using the fusion curve of PCR products, HLA sequences can be matched quickly for all experimental machine manufacturers. Different from the melting temperature (TM), it is only a point on the melting curve. High resolution melting is to analyze the whole melting curve. The shape of fusion curve is used as an indicator to show the sequence matching of heteroduplex formed by heterozygous DNA. After that, the two DNA were mixed in the ratio of 1:1, re fused, and the new curve was compared with the original curve to verify the sequence matching. If the sequences of the two samples are not exactly the same, the shape of the fusion curve of the heteroduplex will change after mixing

olofgidl F of Uppsala University in Sweden has developed HRM analysis of two hypervariable regions HVI and hvii in the mitochondrial genome to distinguish the DNA of different individuals as a pre screening before sequencing in forensic identification. 2. The results show that the sequence variation of HVI and hvii in mitochondrial DNA does produce the difference in the shape of fusion curve, which can distinguish the DNA of different individuals, so as to eliminate mismatched forensic materials, thus saving the time and cost of mitochondrial DNA sequencing. In addition, compared with other technologies, HRM has the advantages of simple operation and low price, and can screen a large number of samples at the same time

mutation scanning

single base change, insertion and deletion can be detected by HRM. In terms of sensitivity and specificity, high-resolution fusion is also higher than traditional methods including DHPLC. When the variant is a low allele fragment, it is even more sensitive than DNA sequencing. Sequencing can only detect as low as 20% of the variants, while HRM can detect as low as 2% of the variants. Moreover, the time, cost and effort spent on sequencing are different from those of HRM

for PCR products below 400bp, the sensitivity and specificity of scanning single base changes were 100%. For 400 to 1000bp PCR products, the sensitivity was 96.1% and the specificity was 99.4%. The position of variation in PCR products did not affect the scanning accuracy. Although HRM is designed to detect heterozygotes (mutations in one allele), it can also detect homozygotes (mutations in both alleles). For hemizygous variants (X-linked or Y-chromosome), it is best to mix unknown samples with samples of known wild-type

lmna gene mutations can lead to a variety of disorders, now known as lamin syndrome. Because there are so many individuals to be studied, it is necessary to use a particularly sensitive and specific method for mutation scanning. Therefore, gillesmillat et al. Developed HRM analysis 3 suitable for LMNA mutation scanning. They used both HRM and DHPLC to screen 64 patients with dilated cardiomyopathy. The results showed that HRM analysis could easily identify any gene variation that could be detected by DHPLC or direct sequencing, and the speed was 2 times faster than DHPLC, which was also more economical. Researchers believe that HRM analysis is a highly sensitive, high-throughput and low-cost method to identify LMNA genetic variation

genotyping

traditional genotyping methods are not only time-consuming and laborious, but also require the purchase of expensive probes. In contrast, HRM analysis has many advantages, without amplification and preparation, eliminating the risk of pollution; Probe money is saved; It can also detect unknown variants, which the probe can do nothing about. With the saturated dye, the PCR product is labeled throughout the whole process, so all fusion regions can be detected. Different heterozygotes in the same amplicon can also be distinguished by the difference of curve shape. HRM can distinguish about 93% of heterozygotes. Most homozygotes can also be distinguished by melting, but not all. About 4% of human single base variants have the same TM and have symmetrical images. In this case, known homozygotes can be mixed with unknown homozygotes for analysis

lassekristensen et al. Developed HRM analysis for key genes involved in methyl metabolism (MTHFR, MTR and DNMT3b), genotyped them, and detected them on rotor-gene6000 and other instruments. 4. They believe that HRM analysis is more economical than genotyping based on TaqMan probe. No probe is required, less optimization is required for analysis, and the success rate is higher. Compared with pyrophosphate sequencing, SNP chip and other technologies, HRM is the best choice

methylation analysis

hrm analysis also provides a new way for the detection of DNA methylation status. DNA samples were treated with bisulfite to convert unmethylated cytosine into uracil. Thus, the original unmethylated template produced a PCR product with a lower TM than the methylated template. HRM can also determine the proportion of methylation in a specific sample. Different proportions of methylated and unmethylated DNA were mixed to make a standard curve, and the melting curve of the sample was compared with it to determine the proportion of methylation in the sample

abnormal methylation patterns are often associated with cancer, so ercismith et al. Developed and verified HRM analysis for rapid quantitative DNA methylation status5. They used mathematical methods to standardize the original fluorescence data generated by heating DNA, and calculated the melting start and end temperatures from these data, so as to reflect the methylation level of template molecules. After that, they also used oligonucleotides with known methylation levels and DNA for verification. They found that the rapid single tube method for quantitative methylation by HRM analysis was reliable, and the primers were easy to design without special software. The obtained parameters provide objective description and quantification of methylation in samples, and can be used for statistical comparison between samples

in conclusion, HRM technology is widely used, simple and economical, accurate and reliable, and suitable for any laboratory

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