Probe: There are many labeling methods, among which isotope labeling and biotin labeling are commonly used. Hybridization methods can be divided into liquid phase hybridization and solid phase hybridization.
Hybridization technology: At present, solid-phase hybridization is widely used. In this method, the single-stranded nucleic acid sample to be detected (if it is double-stranded, it must be denatured into single-stranded) is first bound to the nitrocellulose membrane, and then hybridized with the labeled probe in the solution. By combining electrophoresis and autoradiography, the hybridization map was obtained, and then qualitative or quantitative analysis was carried out. Molecular hybridization is widely used in biochemistry and molecular biology as a means to detect and identify the base sequence of nucleic acid fragments. It has been used in the diagnosis of infectious diseases caused by some viruses or bacteria in the medical field. It can also be used in genetic engineering. The combination process of subunits from different sources of protein can also be called hybridization.
In liquid-phase molecular hybridization, nucleic acid molecules from both sources are in solution and can move freely, and one of them is often labeled with isotopes. The analysis of renaturation kinetics data can reveal the general situation of eukaryotic genome structure, such as the content and distribution of various repetitive sequences. In solid-phase molecular hybridization, one nucleic acid molecule is fixed in an insoluble medium, while the other nucleic acid molecule is in a solution, and the nucleic acid molecules in the two media can contact freely. Commonly used media include nitrocellulose filter membrane, hydroxyapatite column, agar and polyacrylamide gel. In the early days, molecular hybridization with agar as the fixed medium was used to determine the homology of DNA from bacteria to human beings.
(1) solid phase hybridization
Firstly, a nucleic acid chain participating in the reaction is fixed on a solid support, and a reaction nucleic acid is free in the solution. Solid supports include nitrocellulose filter membrane, nylon membrane, latex particles, magnetic beads and microplates. Because of the advantages of solid-phase hybridization, such as easy rinsing and removal of non-hybridized free fragments, easy detection of hybrids left on the membrane and prevention of self-renaturation of target DNA, it is most commonly used. Commonly used types of solid-phase hybridization include colony in situ hybridization, dot hybridization, slit hybridization, Southern blot hybridization, Northern blot hybridization, tissue in situ hybridization and sandwich hybridization.
(2) liquid phase hybridization
The two nucleic acid strands involved in the reaction are both free in solution. A hybrid type with the earliest research and complex operation, although sometimes used in the past 30 years, is not as common as solid phase hybridization. The main disadvantage is that it is difficult to remove the excess unhybridized probes in the solution after hybridization and the error is high. In recent years, due to the continuous improvement of hybridization detection technology and the practical application of commercial gene probe diagnostic kit, the rapid development of liquid phase hybridization technology has been promoted.
Edit this section of solid-phase membrane nucleic acid molecular hybridization (1) colony in situ hybridization.
（Colonyinsituhybridization）
The bacteria were transferred from the culture plate to the nitrocellulose filter membrane, then the colonies on the filter membrane were lysed to release DNA, and then the DNA was dried and fixed on the membrane, and hybridized with the probe labeled with 32P. Autoradiography was used to detect the hybridization signal of the colonies, and it was aligned with the colonies on the plate.
(2) Dot hybridization
（Dotblotting）
The method is to spot the tested specimen on the membrane and bake and fix it. This method takes a short time and can be used for semi-quantitative analysis, and multiple samples can be detected simultaneously on one membrane. In order to make the sampling accurate and convenient, there are many manifolds in the market, such as MinifoldI and II, Bio-Dot(Bio-Rad) and Hybri-Dot, which have many holes. When the sample is added into the holes, it will flow to the membrane in the form of spots or slits under negative pressure. Rinse the injection holes repeatedly, take out the membrane and bake it or irradiate it with ultraviolet rays to fix the sample. At this time, the membrane can be hybridized.
(3)Southern blot hybridization
（Southernblotting）
It is the basic technology to study DNA map, and it is of great value in genetic diagnosis DNA map analysis and PCR product analysis. The basic method is to digest the DNA sample with restriction endonuclease, then separate the enzymolysis fragments by agarose gel electrophoresis, and then transfer the DNA from the gel to nitrocellulose filter membrane (nylon membrane is also used for a long time) by alkali denaturation, neutralization with Tris buffer solution, and then use it for hybridization after drying and fixing. The relative position of DNA fragments in the gel was maintained during the transfer to the filter membrane. The DNA attached to the filter membrane hybridizes with the probe labeled with 32P, and the position of each DNA band complementary to the probe is determined by autoradiography, so that the position and size of DNA fragments containing a specific sequence in many enzymolysis products can be determined.
(4)Northern blot hybridization
（Northernblotting）
DNA imprinting technique was founded by Southern in 1975, and it is called Southern imprinting technique. Northern blotting is called northern blot hybridization because it corresponds to DNA, and protein blotting similar to this principle is called westernblotting. RNA blotting by Northern blot hybridization is similar to DNA blotting by Southern blot hybridization, except that the RNA is denatured by methylmercury oxide, glyoxal or formaldehyde before injection, instead of NaOH, because it will hydrolyze the 2'- hydroxyl group of RNA. After RNA denaturation, it is beneficial to combine with nitrocellulose membrane in the transfer process. It can also be transferred in high salt, but it is not firmly combined with the membrane before baking, so the membrane cannot be washed with low salt buffer after transfer, otherwise RNA will be eluted. EB cannot be added to the gel, because it affects the combination of RNA and nitrocellulose membrane. In order to determine the fragment size, a marker can be added to the same gel for electrophoresis, and then the marker gel is cut off, colored and photographed. The sample glue is transferred by Northern, and the method of coloring the marker glue is to soak it in 0. 1mol/L ammonium acetate 10min containing 5μg/mlEB in a dark room, and then it can be decolored in water. When taking photos with a primary imaging camera under ultraviolet light, the colored RNA glue should be exposed to ultraviolet light as little as possible. If it is exposed to too much ultraviolet light or incandescent lamp for too long, the RNA signal will be reduced. When separating fully functional mRNA from agarose gel, methylmercury hydroxide is a powerful and reversible denaturant, but it is toxic, so many people like to use formaldehyde as denaturant. All operations should avoid RNase pollution.
(5) tissue in situ hybridization
（Tissueinsituhybridization）
In-situ hybridization for short refers to in-situ hybridization of tissues or cells, which is different from in-situ hybridization of colonies. In-situ hybridization of colonies requires cracking bacteria to release DNA and then hybridization. In-situ hybridization, after proper treatment, increases the permeability of cells and allows probes to hybridize with DNA or RNA. Therefore, in situ hybridization can determine the spatial position of the complementary sequence of the probe in the cell, which has important biological and pathological significance. For example, in situ hybridization of dense chromosomal DNA can be used to show the location of specific sequences; The hybridization of nuclear DNA during division can study the functional arrangement of specific sequences in chromatin; Hybridization with cellular RNA can accurately analyze the distribution of any RNA in cells and tissues. In addition, in-situ hybridization is also an important technique to show the distribution and trend of cell subsets and the existence mode and location of pathogenic microorganisms.
The probe used for in-situ hybridization can be single-stranded or double-stranded DNA or RNA probe, and the length of the probe is usually100 ~ 400 nt, if it is too long, the hybridization efficiency will decrease. The recent research results show that oligonucleotide probes (16 ~ 30nt) can freely enter and exit the cell walls of bacteria and tissues, and the hybridization efficiency is obviously higher than that of long probes. Therefore, oligonucleotide probes and asymmetric PCR-labeled small DNA probes or in vitro transcription-labeled RNA probes are the preferred probes for tissue in situ hybridization.