Gene mutation detection technology
Gene mutation detection technology. The main methods currently used for EGFR gene mutation detection include direct sequencing, ARMS, DHPLC, HRM, PCR-SSCP, mutant enrichment PCR, and micro-digital PCR.
Gene mutation detection technology
With the continuous deepening and development of human genomics, pharmacogenomics and tumor molecular biology research, the mode of tumor treatment has gradually shifted from empirical guided treatment to gene-oriented individualized treatment.
In recent years, Epidermal Growth Factor Receptor (EGFR) has become a hot spot in tumor treatment and anti-tumor drug research [EGFR has mutations or amplifications in a variety of tumor cells, and the largest number of mutations has been detected It is Non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (TKI) have been widely used in the treatment of patients with mutant NSCLC. Therefore, the detection of EGFR gene mutations in lung cancer patients has become an urgent need for clinical treatment. ]. The following uses EGFR gene mutation as an example to introduce various technical methods of gene mutation detection.
The main methods currently used for EGFR gene mutation detection include direct sequencing, ARMS, DHPLC, HRM, PCR-SSCP, mutant enrichment PCR, and micro-digital PCR. The following will briefly introduce these detection methods:
1. Direct sequencing method
The direct sequencing method uses the principle of the Sanger sequencing method and is currently recognized as the gold standard for EGFR gene mutation detection. Its advantages are relatively low cost, very intuitive, can directly read the base sequence of DNA, and can detect both known and unknown mutations. The disadvantage is that it needs to amplify, purify, and analyze the sequencing samples. The process is cumbersome, time-consuming, and requires relatively high material and technical requirements, which is harmful to the environment and operators. More importantly, the sensitivity of the direct sequencing method is limited, and the mutation content of the tumor tissue must reach at least 20% before it can be detected by the direct sequencing method.
Pyrosequencing technology is a new generation of DNA sequence analysis technology. Its principle is to add only one dNTP in each round of sequencing reaction. If the dNTP is paired with the template, the polymerase can incorporate it into the newly synthesized DNA. The pyrophosphate group (PPi) is released in the chain. After a series of enzyme cascade chemiluminescence reactions, PPi is converted into a visible light signal, and the intensity of the signal is proportional to the number of nucleotides incorporated in the reaction. Therefore, it is possible to quickly and accurately determine a short target fragment without electrophoresis without fluorescent labeling of DNA fragments. The disadvantage is that it is not as popular as direct sequencing, the sequencing length is short, and it is easy to contaminate.
3. Probe amplification refractory mutation system (ARMS)
The principle is: using Taq DNA polymerase lacks 3’→5′ exonuclease activity, the 3’end base of the PCR primer must be complementary to its template DNA in order to effectively amplify. For different known mutations, design appropriate Primer to detect the mutant gene.
In this method, when designing primers, design a mismatched base at the 3’end of the primer, one complementary to wild DNA and one complementary to mutant DNA, so that it can only complement the mutant or wild type and only amplify the mutant or wild type. Type gene. The amplified PCR products can be quantitatively analyzed by real-time PCR (real-time PCR). At present, two more mature commercial kits have come out, one is the DxS ARMS EGFR detection kit produced by DxS in the United Kingdom, and the other is the Adx ARMS EGFR detection kit produced by some countries’s Xiamen Aide Biomedical Technology Co., Ltd. Both can detect 29 common mutations.
The former uses a scorpion probe, and the latter uses a double loop probe. The ARMS method is more sensitive than the direct sequencing method, and its test results are more consistent with the clinical efficacy. The detection of EGFR mutations in small specimens such as fiberoptic bronchoscopy biopsy and plasma or serum specimens is more suitable for the ARMS method. However, the ARMS method requires special probes and can only detect one mutation at a time.
4. Denaturing high performance liquidchromatography (DHPLC)
DHPLC uses the difference in melting characteristics between mismatched hybrid double-stranded DNA and homozygous double-stranded DNA to separate them. Because the hydrogen bond at the mismatch site of hybrid double-stranded DNA is broken to form “bubbles”, under partial heat denaturation conditions, it is easier to melt to form a Y-shaped structure, and the binding ability to the stationary phase is reduced, so the hybrid The double strands will be eluted before the homozygous double strands, and the difference of the elution peaks can be used to determine whether there are mutations. Compared with the sequencing method, DHPLC is simple, fast, and highly sensitive. It can be used not only for the detection of known mutations, but also for scanning for unknown mutations. Disadvantages of DHPLC: it can only detect mutations, but cannot detect homologous mutations and mutation types. As a result, the interpretation is prone to errors, and when there are multiple fragments to be detected, due to multiple melting temperatures, multi-step detection is required, which increases the workload.
5. High resolution melting curve analysis technology (high resolution melting analysis, HRM)
Utilizing the different melting curves of DNA sequences of different lengths or different base compositions, directly run high-resolution melting after PCR for sample mutation analysis. Using its extremely high temperature uniformity and temperature resolution, the difference of 0.1℃ can be distinguished, so that The resolution accuracy reaches the distinction of single base differences. Each segment of DNA forms a unique melting curve due to its different bases. Like DNA fingerprints, it has high sensitivity, specificity, stability and repeatability. Compared with the sequencing method, it has high sensitivity and can be used for the detection of somatic mutations and the screening of mutations before sequencing. The method does not require sequence-specific probes, and is not limited by mutation base sites and types, is simple to operate, does not require PCR follow-up operations, and has good selectivity. However, this method can only analyze small fragments of DNA with a single purity and requires sufficient templates. Stable and reliable results can be obtained when the template content is not less than 1ng.
6. PCR-Single strand conformation polymorphism analysis (PCR-SSCP)
Single-stranded conformation polymorphism means that single-stranded DNA has a certain spatial conformation due to the base pairing within the strand. When the bases are changed, single-stranded DNA will form a different conformation. In non-denaturing polyacrylamide gel electrophoresis, the mobility of single-stranded DNA is not only related to the length of the DNA, but also mainly depends on the spatial conformation formed by the single-stranded DNA. Single-stranded DNA of the same length has a different sequence or a single base. Difference, the conformation formed will be different. The PCR products are subjected to single-stranded DNA gel electrophoresis after denaturation, and the single strands with base insertions, deletions or substitution mutations will appear different electrophoretic bands. Compared with the direct sequencing method, PCR-SSCP is more sensitive, does not require special equipment, and can also detect unknown mutations, but it also has its limitations: electrophoresis time is longer, the operation steps are more cumbersome, and only qualitative analysis can be performed. Parallel standard controls are greatly affected by experimental conditions and are prone to false negatives.
7. Mutant-enriched PCR (mutant-enriched PCR)
Mutant enrichment PCR is a two-step PCR that uses restriction enzymes to selectively digest the wild-type EGFR gene. After the first PCR, the wild-type EGFR gene is selectively digested to enrich the mutant EGFR gene. Then perform the second PCR, and finally use electrophoresis to detect the PCR product, and determine whether the EGFR gene is mutated according to the nature of the PCR product (size or presence or absence). Compared with the direct sequencing method, because the mutant enrichment PCR has two PCR amplifications, the detection of EGFR activating mutations is more sensitive and specific, and can detect a mutant gene from 103-104 wild-type copies. However, this method also has obvious shortcomings: it requires two PCRs, and requires enzyme digestion, the operation is complicated, time-consuming, and easy to contaminate.
8. Micro-digital PCR (microfluidics digital PCR)
Micro-digital PCR is a digital PCR technology based on integrated fluidic circuits (IFC) chips (currently micro-droplet digital PCR is mainly micro-droplet). The principle is: Dilute and subdivide the sample by a large multiple, until the number of molecules to be tested in each subdivided sample does not exceed 1, and then pass each subdivided sample under the same conditions at the same time after PCR Gene chip counting one by one is an absolute quantitative method. This method has a low probability of contamination, and the sensitivity can reach the level of a DNA strand. It is currently the most sensitive EGFR gene mutation detection method known, and it is especially suitable for the detection of body fluid specimens with low tumor DNA content. Compared with the histological results, the sensitivity and specificity of this method for detecting plasma samples are as high as 92% and 100%, respectively. The disadvantage of this method is that its cost is relatively high and it is not popular at present, and its clinical significance needs to be further verified.
In summary, the current direct sequencing method is still the gold standard for EGFR gene detection, but due to its low sensitivity, it is only used for the detection of tissue samples, which limits clinical applications; the sensitivity of pyrosequencing is 5%- 10%, especially suitable for the detection of known sequences, not only has the intuitive advantages similar to the direct sequencing method, but also allows relative quantification of mutations; the mutant enrichment PCR method requires electrophoresis detection, and the sensitivity does not meet the requirements of detecting ctDNA for EGFR; Although the HRM method has improved automation, it can only analyze small fragments of DNA with a single purity, and requires a template content of not less than 1ng, and the sensitivity does not meet the requirements of EGFR detection for ctDNA; while the sensitivity and specificity of ARMS and micro-digital PCR are higher. High, it can be used to detect the trace free EGFR mutant gene in serum. Compared with other methods, the degree of automation is greatly improved, the time is shorter, and the operation is simpler.
With the continuous emergence and maturity of new technologies such as Scorpion probes and IFC chips, ARMS and micro-digital PCR will present unparalleled advantages in clinical genetic diagnosis.
At present, EGFR mutation detection samples are mainly tissue paraffin sections, but more than 70% of NSCLC patients are in stage IIIB and IV at the time of diagnosis. Most of them have lost the opportunity for surgery and the tumor tissue is difficult to obtain, and these patients often need EGFR -TKI treatment population. Therefore, in recent years, more and more researches have been applied to the detection of EGFR gene in cytology and blood samples, and these specimens often require high sensitivity of detection methods due to the limitation of DNA content. Therefore, the development of more sensitive, specific, simple and easy-to-use mutation detection methods, and simple result determination will have great application prospects in clinical EGFR mutation detection [Currently the market application technology is mostly the ARMS+qPCR method, There is also the NGS method of CAPP-SEQ, and the patented technology as well as the cSMART and FireFly methods. The future will definitely be a very large and promising competitive market].
(source:internet, reference only)