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    Home > Biochemistry News > Biotechnology News > The detection of genetic mutations.

    The detection of genetic mutations.

    • Last Update: 2020-10-26
    • Source: Internet
    • Author: User
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    of genetic mutations has become one of the hot spots in life science research, and the detection method has developed rapidly. The mutation type of human cell carcinoma gene has been described above, for the detection of gene mutation, before 1985, using Southern imprinting method, can screen out the gene loss, insertion and code recombination and other mutation forms.
    for mutations that cannot be detected by using the method, only complex and time-consuming
    . DNA
    sequence determination and analysis.
    polymerase chain reaction (polymerase chain reaction,
    . PCR
    ) technology is the most important progress in mutation research, so that gene mutation detection technology has made great progress, almost all of the molecular diagnostic technology of gene mutation detection is based on PCR, and new methods derived from PCR continue to appear, has reached more than 20 kinds, the degree of automation is also getting higher and higher, analysis time is greatly shortened, the accuracy of the analysis results is also greatly improved.
    include single-stranded polymorphism (single-stranded comformation polymorphism, SSCP) and hetero-duplex analysis (HA). Here are several PCR derivative techniques and classical mutation detection methods, which can be used for reference when selected according to testing purposes and laboratory conditions..PcR-SSCP method PCR-SSCP method is on non-this polyacrylamide gel, short single-stranded DNA and RNA molecules according to their street-based sequence to form different compositions, a base change will affect its composition and cause its movement speed on the gel changes.
    The basic principle is that single-stranded DNA will form a secondary structure under neutral conditions, which depends on its base composition, and even if a base is different, it will form different secondary structures and the same migration rate. Because the method is simple and fast, it is widely used for the detection of unknown gene mutations.
    when using PCR-SSCP to detect PCR products less than 200bp, the mutation detection rate can reach 70%-95%, and when the fragment is greater than 400bp, the detection rate is only about 50%, and the method may have a false positive rate of 1%.
    application of PCR-SSCP method should pay attention to the best conditions of electrophoresis, the general mutation type has no great effect on the sensitivity of detection, and the method can not determine the exact location of the mutation, but also through sequence analysis to determine. Sarkar and others believe that for fragments larger than 200bp, using their RNA molecules as SSCP will improve their recording sensitivity.
    use of PCR-SSCP detection point mutations have been reported in most of the human tumor tissue or cells, such as breast cancer, esophageal cancer, lung cancer, stomach cancer, liver cancer, pancreatic cancer and so on. The genes tested include a variety of cancer genes and anti-cancer genes, but also the most commonly used method to detect the cancer suppression gene p53 mutation, only the detection of 5-8 exons can find more than 85% of the p53 gene mutation. Because the method is simple and fast, it is especially suitable for the screening of gene mutation research in large samples.-origin double-stranded analysis method (HA) HA method directly on the denatured gel to separate the hybridized mutant type of wild DNA double-stranded. Due to mutations and wild-type DNA, heterogenous hybrid double-stranded DNA forms a protrusion at its misalignment, and in non-denatured gel electrophoresis, a different migration rate is produced than the corresponding iso-dual DNA.
    is similar to SSCP, the difference is that SSCP is separated by single-stranded DNA, HA method is separated by double-stranded DNA, but also suitable for small fragment analysis. However, HA complements some mutations that cannot be detected with SSCP, and the combination of the two can increase the detection rate of mutations to nearly 100%.the mutant-enriched PCR Method This method is based on the use of a known restricted endoenzyme site at a cocoon site in the ras gene family, such as the BstNI site of the K-ras gene's 12th cocoon, and the 13th secret Cuban sub has a BgI.II. site.
    with a chain-renewed second nest PCR to amplification including K-ras 12th, 13th colicoma DNA fragments, between the two amplification reactions with the corresponding endoenzyme digestion amplification of DNA fragments, wild type because of enzymatic cutting can not enter the second PCR amplification, and mutants can fully enter the second PCR amplification and obtain the rich collection of the product.denaturation gradient gel electrophoresis (denaturing gradinent electrophoresis, DGGE) DGGE method to analyze PCR products, if the mutation occurs in the dna region of the first unchain, detection rate of up to 100%, detection fragments up to 1kb, the most suitable circumference of 100bp-500bp.the basic principle of
    is based on the fact that when double-stranded DNA is carried out in a denatured gradient gel to a gel position consistent with DNA denaturation humidity, dna is partially unchained, electrophoresis is reduced, and when there is a base change in the DNA strand of the delineation chain, the unchain occurs at different times and is separated due to the process that affects the change in electrophoresis speed.
    Since this method is to use temperature and gradient gel migration rate to detect, a set of special electrophoresis device is required, the synthetic PCR quotient is best added at the end of 5' a 40bp-50bp GC clip to help detect mutations occurring in the high melting point area.
    on the basis of DGGE, the TGGE method (temperature gradient gel electrophoresis gelelectrophoresisis, TGGE) has been developed to replace chemical denaturation agents with humidity gradients. Both DGGE and TGGE have commercialized electrophoresis devices, which, once established, are simple to operate and suitable for the detection and screening of large samples.chemical cutting mismatch (CCM) CCM is a technique developed to detect mutations based on Maxim-Gilbert sequencing, which detects mutations with similar accuracy to DNA sequencing.
    The basic principle is to mix the DNA fragments to be tested with the corresponding wild DNA fragments or DNA and RNA fragments, in heterogenous hybrid double-stranded nucleic acid molecules, misaligned C can be cut by serotonin or pyridine, misaligned T can be cut by four oxidation hungry, by denatured gel electrophoresis can determine whether there is a mutation.
    this method detection rate is very high, is also the longest detection fragment method, has been reported to detect 1.7kb fragment, if the positive and antiseth chain analysis, detection rate of up to 100%. Fluorescence detection systems enhance sensitivity and detect 1 mutant cell out of 10 cells.
    the
    chemical reagents
    in this method are toxic and have developed a carbon diamine test (catodiimide, CDI), a non-toxic substance that can also detect point mutations in large pieces of DNA.gene-specific oligonucleotide (ASO) ASO is a hybrid-based detection technique for known mutations. Using a combination of PCR and ASO, a fragment of oligonucleotides of about 20bp is designed, which contains the site where the mutation occurs, as a probe, and interbreeds with the DNA of the PCR-pulled sample fixed to the membrane.
    can use various types of oligonucleotide probe, while the wild type probe as a control, if there is a positive hybrid belt, then there is a point mutation corresponding to the ASO probe in the table canal sample, ASO needs to strictly control the hybridization conditions and set standard control to avoid false positive and false negative. At present, some cancer gene ASO mutations have been detected in commercialized test boxes..DNA chip technology (DNA chip) DNA chip technology is a new DNA analysis technology developed after the 1990s, it brings together integrated circuit computers, laser concentr scanning, fluorescent marker probes and DNA synthesis and other advanced technologies. It can be used for gene positioning, DNA sequencing, physical map and genetic map construction.
    There is also broad prospects for DNA chips in gene mutation detection, based on the principle that many known sequences of oligonucleotide DNA are arranged on an integrated circuit board, overlapping one base with each other, and covering all the genes required for detection, and interbreeding the normal and mutated DNA of fluorescent markers with two DNA chips, due to the presence of at least two DNA chips. 1 base difference, normal and mutated DNA will get different hybrid map, after a total of
    microscope
    to detect the fluorescent signals produced by two DNA molecules, can determine whether there is a mutation, the method is fast and simple, high degree of chip dynamics, has great potential for development, will play a very important role in the gene mutation detection center.the ligase chain reaction (ligase chain reaction, LCR) compared with other nucleic acid amplification techniques, the greatest feature is the ability to accurately distinguish individual gene mutations in the gene sequence, first applied by Landegree in 1988 to the molecular diagnosis of sickle cell anemia.
    LCR is based on the DNA connecting enzyme to connect 5'-phosphoric acid from one DNA strand to another adjacent chain 3'-hydroxyl, using two pairs of complementary citants, two pairs of DNA denatured by
    heating
    Complementary, under the role of the connecting enzyme, so that the adjacent two citations of 5'-phosphoric acid and 3'-hydroxyl form a phosphate deester bond and connection, the previous connection product as the template for the next cycle reaction, if there is a mutation in the paired base can not be connected and amplified.
    LCR product detection was initially identified by the 32p mark upstream lead 3' non-end, after the separation of the denatured gel electrophoresis radiation self-development, its detection sensitivity reached 200 target molecules. A detection probe spanning two citations can also be designed to interbreed with LCR products. In recent years, non-nuclein markers such as luciferin and geogosin have been applied.
    Batt developed a simpler approach in 1994,
    the microplate
    sandwich hybridization. Because of LCR's fast, egg-specific and sensitive properties, as well as its ability to detect individual base mutations, it is used in molecular diagnosis of tumor gene mutations and combined with PCR to increase their sensitivity.Established in 1989, allele-specific amplification (ASA) ASA is a development of PCR technology applications, also known as amplification block mutation systems (amplification refractory mutation system, ARMS), allele-specific PCR (ASPCR), etc., for the detection of known mutation genes.
    This method by designing two 5' end quotations, one complementary to normal DNA, one complementary to mutant DNA, for purely combined mutations, two parallel PCRs are added to these two citations and 3' end-of-the-line quotations, respectively, to absorb the results complementary to the mutant DNA to extend and obtain PCR amplification. If the misalmation is located at the 3' end of the quotation, causing the PCR to not extend, it is called ARMS.
    ARMS and ASPCR, using the principle of multiple PCR, can detect two or more allegen mutation points in the same system at the same time. The detection rate of the ASA method depends on the optimization of the reaction conditions and the mismatch extension of the possible mismatch between the lead and the target DNA, especially when the mismatched base is G:T, when the experimental conditions such as the target DNA of the lead are adjusted, the concentration of Taq DNA polymerase, etc. can be highly specific.
    addition of methamide to the reaction system can also reduce nonse specific amplification. Error extension can also be prevented by introducing a misalmed base at the second base at the 3' end of the profile, creating a double misalm match between it and the template. . RNase A cleavage Under certain conditions, the amino source double-stranded nucleic acid molecular RNA: RNA or RNA: mismatched bases in DNA can be cut by RNaseA, and the cutting products can be electrophoresis by denatured gels. RNaseA's cutting efficiency at the misalide is low, not even when the misaltered base is nirconium, and when the misalquisition base is niche, the cutting efficiency is high.
    if only one chain of dna is analyzed, the mutation detection rate is only 30%, such as the analysis of both just and antisant chains, the detection rate can reach 70%. This method requires the preparation of RNA probes, which adds complexity to the operation, but can be used for detection of large fragments of 1-2kb and to determine mutation points. With these advantages, it is still used as a classical method for analyzing unknown mutations. more than 150 years after the discovery of chromosomes, chromosomal testing has been widely used in cytogenetic research in animals, plants, and humans, with the development of chromosomal technology and
    molecular biology
    technology. The scope of chromosomal research is also expanding, especially for the diagnosis of tumor molecules.
    chromosomal changes in tumor cells are a very common phenomenon, can be divided into primary and secondary two categories. In terms of the biological basis of tumor formation, primary chromosomal changes are related to the direct causes of tumors, and various forms of chromosomal distortion can be found in tumor cells, such as missing, repeating, altering, rearm, monosome fracture and in-nucleus replication.
    detection of chromosomes is of great significance to the diagnosis of tumors, the identification of diagnosis, the identification of biological behavior and so on. Chromosome detection methods are progressing rapidly, and the accuracy of detection is constantly improving, mainly introducing fluorescence in-place hybridization and PRINS methods. fluorescent in-place hybridization technology (fluorescen).
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