Molecular Hybridization

Molecular hybridization is a technique for qualitative and quantitative analysis of nucleic acid or protein using the principle of specific binding between molecules, mainly including nucleic acid hybridization and protein hybridization. Nucleic acid molecular hybridization is an experimental technique established on the basis of nucleic acid denaturation and renaturation. It is a process in which two single nucleotide strands with certain homologous sequences form heteroduplexes under annealing conditions according to the principle of complementary base pairing. Protein hybridization is a biotechnology based on antigen-antibody specific combination.

Procedure

Pre-hybridize
Use non-specific DNA molecules and other macromolecular compounds (blocking agents) to block non-specific sites in the membrane to be hybridized, thereby reducing hybridization background.
Mark the probe
There are many methods for labeling DNA or RNA in vitro, such as: end labeling, random primer labeling, nick translation, in vitro transcription and various PCRs.
Hybridize
The labeled probe is added to the hybridization solution, and under certain (temperature) conditions, the probe is hybridized with the DNA on the membrane.
Wash the membrane
Use solutions with different compositions and ionic strengths to wash away the blocking agent and non-specifically hybridized probes on the membrane.
Wrap the membrane
Remove the membrane from the washing solution, dry it on filter paper until no water film is visible on the surface of the membrane, wrap it with plastic wrap, press X-ray film, and place it at -20 or -70°C for 3-7 days.
Develop x-ray film
Take out the X-ray film under the red light in the dark room, put it into the developing solution until the hybridization band appears, and then rinse it in clean water. Then put it into the fixer and fix it until clear. After rinsing with tap water, let dry, and read the slides.

Types

In situ hybridization
There are two types of in situ molecular hybridization, one is slide in situ hybridization, and the other is membrane in situ hybridization. Slide in situ hybridization is used for gene localization of chromosomes, or to observe the distribution of different RNAs in tissues. Molecular hybridization on the membrane is to replicate colonies or plaques on nylon membranes, which only absorb single-stranded DNA. This method is often used to catch genes from gene libraries.
Dot blotting
Dot blotting is to spot the sample to be tested on the film, and bake it to fix it. This method takes a short time and can be used for semi-quantitative analysis, and multiple samples can be detected on one membrane at the same time.
Southern blotting
Southern blotting is a basic technique for studying DNA profiles, and it is of great value in DNA profile analysis of genetic diagnosis and PCR product analysis.
Northern blotting
Northern blotting is used to test RNA. The RNA blotting method for Northern blot hybridization is similar to the DNA blot method for Southern blot hybridization. This method can be used to measure the spatiotemporal specificity of gene expression.
Western blotting
Western blotting is used to detect proteins. This method transfers the protein band on the electrophoresis gel of polyacrylamide to the nylon membrane by the action of an electric field, and detects the protein with an affinity reaction, an immune reaction or a binding reaction.

Applications

For locating and detecting specific genes in the genome
For finding the desired gene from the gene library
For genetic diagnosis of genetic diseases, cancer and so on

Hybridization Oven

Hybridization ovens are convection ovens used for molecular assays. It is used in molecular biology labs, research labs, and other labs where DNA, RNA, or protein identification needs to be done. Southern, northern, and western blots all benefit from using a hybridization oven. The constant rotation of the plates inside the hybridization oven means that the probes will be distributed evenly over the membranes, leading to a more reliable result.

Water Bath

A water bath is laboratory equipment made from a container filled with heated water. It is used to incubate samples in water at a constant temperature over a long period of time. Most water baths have a digital or an analogue interface to allow users to set a desired temperature. A laboratory water bath is a preferred heat source for heating flammable chemicals instead of an open flame to prevent ignition.

Centrifuge

A centrifuge is any device that applies a sustained centrifugal force—that is, a force due to rotation. The widest use of centrifuges is for the concentration and purification of materials in suspension or dissolved in fluids. Suspended particles denser than the suspending liquid tend to migrate toward the periphery, while those less dense move toward the centre.

Electrophoresis System

Electrophoresis equipment applies an electric charge to molecules, causing them to migrate towards their oppositely charged electrode. The technique is found in all research and clinical laboratories utilizing DNA and protein applications, and is divided into gel and capillary techniques. Choice of electrophoresis equipment depends on the molecule of study, appropriate method of separation and downstream application.

Laboratory Oven

A laboratory oven is a heating device that meets the precise temperature control and temperature uniformity requirements of laboratory work. It is a standard piece of equipment in many labs, used for a wide range of applications such as drying, evaporating, or curing. Depending on the requirements, laboratory ovens vary in size and volume as well the maximum temperature they can reach.

STEMart provides you with a variety of molecular hybridization technology equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for molecular hybridization, please feel free to contact us.