SNP detection for HRM technology services (best solution for snp detection)
Single Nucleotide Polymorphisms (SNPs) refer to changes in single nucleotide bases, including substitutions, translocations, deletions, and insertions, resulting in polymorphisms in nucleic acid sequences. In the nucleotide sequence of the same chromosome or the same site of different individuals, most of the nucleotide sequences are identical and only one base is different. This is the SNP. SNPs are more abundant in the human genome and occur more frequently, so they are considered to be a new generation of genetic markers following microsatellites.
HRM detection SNP technology is based on denaturation of PCR-amplified products over a range of temperatures, during which time the fluorescence signal in the system is detected in real time. The fluorescence value can be plotted as a function of temperature. Each piece of DNA has its own unique sequence, and thus has a unique melting curve shape, like DNA fingerprinting, with high specificity, stability and repeatability. According to the curve, wild type homozygotes, heterozygotes and mutant homozygotes are accurately distinguished (below).
There are many ways to detect SNPs. Methods with lower cost and greater throughput are mostly limited to known, specific sites, such as the Taqman probe method. Both the analysis of known sites and the search for unknown sites are generally carried out by PCR+sequencing, which is costly and complicated.
As a new generation of genetic scanning tools, HRM technology is a low-cost, high-throughput, fast, and site-independent detection method that is the best choice for SNP screening. It has the advantages that other technologies such as "simple, effective, fast, and inexpensive" are incomparable:
Jane: No sequence-specific probes, no base sites, and detection of known or unknown mutations, SNPs, and methylation sites: closed-tube operations to avoid cross-contamination.
Efficacy: The minimum detection of 0.1%-0,01% of mutations or abnormal methylation detection SNP sensitivity and specificity are 100%.
Fast: No more than 384 samples at a time, complete the test within 60-90 minutes.
Integrity: Simplify the operation steps, reduce labor and reagent costs, and cost far less than sequencing, Taqman probes and other methods.
HRM technology is especially suitable for SNP, mutation or methylation analysis with large sample size and few detection sites. It is a high-throughput method different from gene chip detection method (more detection sites and fewer samples). At the same time, it is also the best way to verify multiple genes after gene chip screening in more samples.
Scheduled SNP related research methods:
| Research methods | advantage | Disadvantage |
Direct sequencing | Gold standard for SNP analysis to identify known and unknown SNP sites | Each site needs to be amplified by PCR and then sequenced. The cost is high, the workload is large, the cycle is particularly long, and the price is expensive. It is not suitable for large-scale analysis of disease association and is easy to cross-contamination. |
Taqman probe method (quantitative PCR ) | Suitable for detection of known SNP sites, small number of sites and high throughput | Expensive (high probe synthesis cost), can not find unknown SNP sites at the same time |
Non-probe common PCR method | The technology is simple and the price is cheap. It only applies to the known SNP judgment. It is impossible to determine which SNP , and it is suitable for laboratory testing of a small number of samples. | Time-consuming and laborious, slower speed and poor sensitivity; RFLP can only detect SNPs with enzyme cleavage sites, and no enzyme cleavage sites can not be detected; all of the above methods require gel electrophoresis, and the secondary structure of DNA strands is likely to cause artificial pseudophases, resulting in results. There is a deviation. SSCP and DGGE take a long time, have poor stability, and have limited sensitivity, making it difficult to work on a large scale. |
Chip technology | Compared with traditional instrument detection methods, it has high throughput, miniaturization, automation, and pollution prevention. | It is only suitable for whole-genome SNP scanning. It is not suitable for SNP detection of single gene, and its precision is low and expensive. |
Illumina technology | This approach allows researchers to detect multiple regions of interest while benefiting from the low initial sample size requirements to detect rare genetic mutations. | High-throughput SNP detection for the scanning analysis of SNPs across the genome is expensive. |
MALDI-TOF MS mass spectrometry | Fast detection, theoretically separate single base changes | |
HRM technology based on Roche LightCyclerTM 480 | High throughput, simple, fast, cost-effective, high sensitivity; closed-tube detection to avoid false positives caused by contamination; both known and unknown SNPs in the amplification zone can be detected; sensitivity and accuracy reach nearly 100% ; HRM analysis and PCR is performed at the same time, no additional instrument is required, and additional instruments are reduced compared to MS, etc., which is undoubtedly less costly, non-specific and more accurate. | Must use Roche LightCyclerTM 480 PCR , or LightScannerTM |
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