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nucleic acid molecular hybridization technology
Because of the high specificity of nucleic acid molecular hybridization and the sensitivity of detection methods, it has become the most commonly used basic technology in molecular biology, and has been widely used in the screening of gene cloning, the production of enzyme cutting maps, quantitative and qualitative analysis of gene sequences and the detection of genetic mutations. The basic principle is that the original nucleic acid single chain with certain ogeneity can be formed into a double chain according to the base complementary under certain conditions (suitable greenhouse degree and ion strength, etc.).
hybridization is between the nucleic acid sequence to be tested and the probe, the nucleic acid sequence to be cloned gene segments, or the unclonalized" > DNA and cell total RNA. Nucleic acid probes are known DNA or RNA fragments marked with radionuclides, biotin, or other active substances that can be specifically complementary to a particular nucleic acid sequence. According to its source and nature can be divided into cDNA probe, genome probe, oligonucleotide probe, RNA probe and so on.
solid-phase hybridization
solid-phase hybridization is the fixation of denatured DNA to a solid substate (cellulose nitrate membrane or nylon membrane) and then hybridization with the probe, so it is also known as membrane imprinting hybridization.
dot hybridization
is the dna or RNA denaturation that is first measured and then fixed to the membrane and then interbred with an excessive amount of marked DNA or RNA probes. The method is characterized by simple operation, prior use of restrictive endoenzyme digestion or gel electro-permanent separation of nucleic acid samples, can be carried out on the same membrane at the same time multiple samples of testing; The disadvantage of this method is that the relative molecular quality of the gene under test cannot be identified, and the specificity is poor, with a certain percentage of false positives.
printing hybridization
Southern imprinting hybridization: Gel ionizes DNA fragments digested by restrictive endoenzymes, denatures DNA on the gel and transfers single-stranded DNA fragments to nitroculose membranes or other solid-phase supports in place, is fixed by dry roasting, and then reacts with the corresponding structure's labeled probe at that time, using radioactive self-development or enzyme reaction to color, to detect the content of a specific size molecule. Enzyme mapping analysis of cloned genes, qualitative and quantitative analysis of genomic genes, analysis of gene mutations and restrictive length polymorphism analysis (RELP) can be carried out.
Northern imprint hybridization: evolved by Southerm imprint hybridization, whose sample is RNA. After formaldehyde or polyacetaldehyde degeneration and electrophoresis separation, transferred to solid phase support, hybridization reaction, to identify the amount and size of specific mRNA molecules in the base. This method is a common method for studying gene expression, which can be used to promote the expression of cancer genes.
differential hybridization
The recombinant phage DNA of the genomic library is transferred to the cellulose nitrate membrane, where two different cDNA probes (e.g., mRNA reversed cDNA from metastatic and non-metastatic cancer tissue) are used to interbreed with DNA on the membrane, analyzing the corresponding location hybridization information on the two membranes to isolate the genes expressed by difference. The false positive rate is lower when compared with the expression genes of less complex hypoxic organisms such as yeast. However, nucleic acid organisms (e.g. human) with very complex genomes are less valuable because of the heavy workload and the low percentage of sequences expressed (only about 5 per cent).
cDNA microray hybridization
A cDNA clone or cDNA PCR product is scheduled in a high array and combined with solid-phase supports (e.g., nylon membranes or active slides) in microscopes, and then hybridized with a mixture of different DNA probes and DNA from the microscope. The hybrid information on the microscope is scanned using < fluorescence, chemical luminescence, > a href" microscope and other technologies. It is more efficient, fast and low cost than differential hybridization technology, and is suitable for large-scale analysis. It has become a commodity. The disadvantage is that it is impossible to overcome the interference of conservative consoquent sequences and resequencing on hybrid information.
oligonucleotide microarray hybridization >
is an in-place synthesis of oligonucleotides on special solid-phase supports, so that they are co-priced to the surface of the support and interbreeded with mixed RNA or cDNA probes with an average length of 20-50nt to improve the specificity and sensitivity of hybridization. A confocus microscope can be applied to detect hybrid information spanning three orders of magnitude. Suitable for the detection of low-abundance mRNA to distinguish the expression characteristics of different members of the gene family, or to identify selective shearing of the same transcription in different tissues and cells. However, there are weaknesses such as greater workload, higher cost and slower speed.
liquid phase hybridization >
refers to the denatured nucleic acid single chain and radiation nuclein marker known acid single chain (probe) in the solution insulation, so that it forms a hybrid complex. After the reaction, the amount of nucleic acid measured can be calculated by separating the unbreeded single chain from the hybrid double chain by hydroxyphosphate or enzymatic solution.
decreasing hybridization (subtractive hybridizatio
is the extraction of mRNA (or reverse-recorded cDNA) from tissue cells of two consistent and different functions, and the liquid hybridization of the tested single-stranded cDNA or mRNA under certain conditions, with excessive driving mRNA or cDNA, and the removal of the hybrids of the same origin through hydroxyphosphate column layer analysis. After multiple rounds of hybridization, the same genetic components between the two are removed, and the specific expression of the purpose gene or industrial gene fragments are retained. Later, the user screens the cDNA library to obtain the full-length sequence of the specific expression of the purpose gene cDNA.
nucleic acid in-
uses a specific labeled known sequence of nucleic acids as a method for complex hybridization and detection of nucleic acids in cell-level or tissue slices, known as nucleic acid in sit. It is used to detect the distribution of DNA on the nucleus or dyeing rest, to interbreed with in-cell RNA to study the expression of water leap by specific genes in the cells of the tissue, and to study whether there are specific bacteria and viruses in cells and tissues. the
method has the advantage of being highly specific and precisely located, being able to study a single cell in a complex tissue without being affected by other components in the same tissue, not requiring the extraction of nucleic acids from the tissue, having a high sensitivity to very low-content target sequences in the tissue, and maintaining the tissue and cell morphology intact. Therefore, it is widely used in medical biology research, such as gene positioning, gene loss, gene change, specific gene integration department of the detection and other research. In recent years, from qualitative development to quantitative, the method is more perfect.
DNA molecular cloning technology (also known as gene cloning technology)
connects DNA molecular fragments to vector DNA fragments in-body, transferring DNA molecular clones (or clones) to cells for mass copying. The basic steps include: the preparation of the purpose gene → the purpose gene and the vector with restrictive endoenzyme cutting and connection, dna recombination→ imported into the host cell →cessing, identification→ amplification and expression.
< p style is "text-align:left;" > vectors (vecors) replicate themselves within cells and lead to the co-proliferation of recombined molecular fragments, resulting in a large number of DNA molecular fragments. The main goal is to obtain a large number of copies of a gene or NDA fragment, with which the exact same molecular clones as the parent molecule allow for an in-depth analysis of the structure and function of the gene, and with the introduction of DNA fragments, there are two DNA libraries, one genomic library and the other cDNA library.bacterial prosites are DNA of a small double-stranded ring structure outside the bacterial chromosome, with a molecular size of 1-20kb, which has a certain effect on certain metabolic activity and drug resistance esotypes of bacteria. The prosite carrier is made of artificial modification and stitching on the basis of natural granules. The most commonly used prosury is pBR322.
phage (phaeg) phage is a virus infected with bacteria, according to its life cycle is divided into soluble type and dissolved prototype two types. The phage vector modified and constructed with wild phage is linear double-stranded DNA, the genome is about 50kb, and the most commonly used mycobacteria is radon DNA and its derivative series.
-6kb of cosmid.
expression vector connects the above-mentioned bacterial particle vector or phage vector to the gene sequence of the initiating gene and polyurucleosome to form the expression carrier.
gene bank construction
A recombined DNA clone group containing random fragments of all the base calendars of an organism, the recombinations of which contain photoreceptory gene fragments, which can be screened by methods such as interbreeding recombination of recombinations in the gene pool by marking the probe, and the resulting grams.