Peculiarities. Southern blotting in molecular genetics. Features In vivo DNA cloning

A specific gene involves the ability to distinguish specific segments of DNA or RNA molecules corresponding to that gene from among the many other segments of DNA or RNA molecules present in a sample of cells or tissue. When genomic DNA is analyzed, the challenge is to detect and study the specific DNA fragment of interest in a complex mixture containing several million fragments obtained by digestion of genomic DNA with restriction enzymes. With RNA samples, the challenge is to detect and measure the volume and quality of a particular copy of mRNA in the total RNA of a tissue in which the mRNA of interest is less than 1 in 1000 of the total number of RNA copies.

Solution Problems detecting one rare sequence among many others involves the use of gel electrophoresis to separate DNA or RNA molecules by size, followed by hybridization with a nucleotide probe that identifies the molecule of interest.

Southern blotting technique(Southern blotting) allows you to detect and study at a rough level many fragments of interest in a seemingly uninformative collection of about a million fragments obtained after processing DNA with restriction enzymes. Southern blotting, developed in the mid-1970s, has become the standard method for examining specific fragments of DNA that have been digested by restriction enzymes. This procedure first involves extracting DNA from an available source. Any cell in the body can be used as a source of DNA, with the exception of mature red blood cells that do not have a nucleus.

During sample analysis DNA The patient usually uses the genomic DNA of lymphocytes obtained by routine vein puncture. 10 ml of peripheral blood contains approximately 108 leukocytes or about 100 micrograms of DNA - a dose sufficient for digestion by restriction enzymes. Genomic DNA can also be obtained from other tissues, including cultured skin fibroblasts, amniotic cells or chorionic villi, which are used for prenatal diagnosis, or from a biopsy sample of tissue from various organs (eg, liver, kidney, placenta).

Trying with millions of different fragments DNA, generated by enzymatic digestion of a genomic DNA sample with restriction enzymes, are applied to the surface of an agarose gel at the starting point. The DNA fragments are then separated by size using agarose gel electrophoresis, in which small particles move faster than larger ones in an electric field. When the DNA of interest, thus separated, is stained with a fluorescent dye such as ethidium bromide, the DNA fragments appear as fluorescent regions distributed along the lane in the gel, smaller fragments above, larger ones below. DNA appears in the gel as a smear rather than as discrete bands because there are usually too many different-sized DNA fragments to separate one from another.

Spot of double-stranded fragments DNA first denatured with a strong alkali to separate the DNA strands. The resulting single-stranded DNA molecules are then transferred from the gel to porous paper filters (hence the second name of the method - “Southern transfer”).

For identification one or more fragments of interest among millions of others, the filter is incubated with a complementary single-strand labeled probe under mating-promoting conditions double molecules DNA. Unbound probes are removed by washing, then the filter with bound radioactive probes is exposed to photographic film, which reveals the position of one or more fragments to which the probe has attached. Thus, specific radioactive bands for each human DNA fragment of interest on the original gel can be seen on the film.

Ability Southern blot identification of mutations is limited because probes can only detect defects that have a measurable effect on restriction fragment size, such as large deletions or insertions. A mutation that changes one or more bases cannot be detected unless it creates or destroys a restriction enzyme recognition site, resulting in a significant change in the size of the fragment detected by the probe. However, there are many methods other than Southern blotting to detect mutations affecting one or more base pairs in a gene.

Southern blot) is a method used in molecular biology to identify a specific DNA sequence in a sample. The Southern blotting method combines agarose gel electrophoresis to fractionate DNA with techniques for transferring length-separated DNA to a membrane filter for hybridization. The method is named after its inventor, English biologist Edwin Southern.

Other transfer (blotting) technologies, such as Western blotting, Northern blotting, Southern blotting, use similar methods, but to detect RNA or protein in a sample, and are named after the method invented by Southern. Since Southern blot is named after the scientist, the term is written with a capital letter, while Western blot and Northern blot are written with a lowercase letter.

Method

Restriction endonucleases cut high molecular weight DNA into smaller fragments.
The DNA fragments are electrophoresed on an agarose gel for length separation.
In case some DNA fragments are longer than 15 kb, before transfer the gel is treated, for example, with hydrochloric acid, which causes depurination of the DNA and facilitates transfer to the membrane.
When using the alkaline transfer method, the agarose gel is placed in an alkaline solution, which denatures the DNA double helix and facilitates the binding of the negatively charged DNA to the positively charged membrane for further hybridization. In this case, the remaining RNA is also destroyed.
A sheet of nitrocellulose (or nylon) membrane is placed on top or bottom of the agarose gel. Pressure is applied directly to the gel or through several layers of paper. For successful transfer, tight contact between the gel and the membrane is necessary. The buffer is transported by capillary forces from an area of high water content to an area of low water content (membrane). This involves transferring DNA from the gel to the membrane. Polyanionic DNA binds to the positively charged membrane through ion exchange interactions.
To finally fix the DNA on the membrane, the latter is heated in a vacuum to a temperature of 80 °C for two hours or illuminated with ultraviolet radiation (in the case of nylon membranes).
Hybridization of a radioactively (fluorescently) labeled sample with a known DNA sequence is carried out with a membrane.
After hybridization, the excess sample is washed off the membrane and the hybridization products are visualized by autoradiography (in the case of a radioactive sample) or the color of the membrane is assessed (in the case of chromogenic staining).

results

Hybridization of a sample with a specific DNA region fixed to the membrane indicates the presence of the analyzed nucleotide sequence in the sample.

Application

Southern blotting, which is performed with genomic DNA treated with restriction endonucleases, can be used to determine gene copy numbers in the genome. A probe that hybridizes only to a single DNA fragment that has not been cut by restriction enzymes will produce a single band on a Southern blot, while multiple bands on the blot indicate that the probe has hybridized to multiple identical sequences. Changes in hybridization conditions (increasing the temperature at which hybridization is carried out, changing salt concentration) lead to an increase in specificity and a decrease in hybridization with close, but not identical, sequences.

Notes

Southern blotting- Southern Blotting Southern blotting of DNA sample. Southern blotting involves electrophoretic separation of DNA and methods of transferring DNA fragments from an agarose gel to a membrane under the influence of electric field for further analysis with... ... Explanatory English-Russian dictionary on nanotechnology. - M.

Southern blotting, Southern transfer Southern blotting, Southern blotting. A method for detecting specific nucleotide sequences by transferring electrophretically separated DNA fragments from an agarose gel onto a nitrocellulose... ... Molecular biology and genetics. Dictionary.

SOUTHERN BLOTTING- (Southern blot analysis) a method for identifying specific forms of DNA in cells. DNA molecules are removed from cells and separated into small fragments using restriction enzymes. These fragments are separated from each other, and with the help of gene... ... Explanatory dictionary of medicine

Southern blot C blot hybridization- Southern blotting, blot hybridization according to S. * Southern blotting, blot hybridization according to S. * Southern blotting or S. hybridization or S. transfer gel blotting technique (see), in which DNA fragments are separated by size in an agarose gel... ...

A method for identifying specific forms of DNA in cells. DNA molecules are removed from cells and separated into small fragments using restriction enzymes. These fragments are separated from each other, and a search is made using a gene probe... ... Medical terms

Southern blotting Southern blotting is a method used in molecular biology to detect a specific DNA sequence in a sample. The Southern blotting method combines agarose gel electrophoresis to... ... Wikipedia

- (from English Blot) common name molecular biology methods for transferring certain proteins or nucleic acids from a solution containing many other molecules onto some kind of carrier (nitrocellulose membrane, PVDF or ... ... Wikipedia

Blotting transfer blotting- Blotting, blotting transfer * blotting, blotting transfer * blotting or blot transfer procedure for transferring electrophoretically separated DNA (see), DNA fragments, RNA, RNA fragments or proteins from a gel (agarose or polyacrylamide) to ... ... Genetics. encyclopedic Dictionary

To identify a gene, the DNA molecule of the genome is split using restriction enzymes into pieces of approximately 15-20 thousand nucleotide pairs in size. The genome split in this way is subjected to electrophoretic fractionation in an agarose gel. The DNA fractions are then denatured by heat and transferred from the agarose gel to a nitrocellulose filter, where they are immobilized. The process of DNA transfer resembles getting wet (in English - blotting) and is called a method Southern blotting. The essence of blotting is that an agarose gel is placed on filter paper soaked in a concentrated saline solution; a nitrocellulose filter is then placed over the gel and dry filter paper is placed on top. The saline solution is absorbed into the dry paper; for this to happen, it must pass through an agarose gel and then through a nitrocellulose filter. The DNA is transferred with the solution, but is retained by the nitrocellulose. DNA immobilized in this way can be hybridize in place with a radioactive probe. Only fragments complementary to it will hybridize with a specific probe. Since the probe is radioactive, hybridization can be detected using autoradiography. Each complementary sequence appears as a radioactive band, the location of which is determined by the size of the DNA fragment. A diagram of the Southern blotting method is shown in Fig. 12.4.

Rice. 12.4. Southern blotting scheme: cleavage of genome DNA using restriction enzymes into pieces of 15,000-20,000 nucleotide pairs; electrophoretic separation of these restriction enzymes in an agarose gel, transferring them to a nitrocellulose filter, hybridization with a DNA probe and detection of the resulting hybrid molecules by autoradiography; *) transfer (blotting) scheme.

The blotting method is highly sensitive and accurate and is widely used in forensics, medicine, and veterinary medicine. Currently, the molecular hybridization method has been developed for the diagnosis of infectious diseases of farm animals, for example, to detect the causative agent of anthrax, brucellosis, tuberculosis, foot-and-mouth disease, swine fever, bird plague, enteroviruses, etc. This method is promising for studying the breeding qualities of animals. It has advantages over the method of studying protein polymorphism markers currently accepted in breeding.

It is believed that the DNA hybridization method can be successfully used in bull selection, since the bull genome can be divided into genes, which are subsequently detected by blotting. In this case, it is necessary to isolate about 75 DNA fragments to evaluate the genome for milk production.

IN last years is being developed new method DNA analysis, so-called “genomic fingerprinting”. Genomic fingerprinting includes the following stages: DNA isolation, fragmentation using restriction enzymes, fractionation using gel electrophoresis. DNA fragments containing hypervariable regions are detected using a special probe - "Jeffreys samples" to which they bind by hybridization. Areas of hybridization are identified by autoradiography.

Research has shown that in this technique, DNA isolated from the Ml3 bacteriophage can be used as a radioactive probe. The DNA of this bacteriophage contains another type of hypervariable sequence, which is also found in the human genome. The principle of the structure of this hypervariable sequence in general outline similar to the structure of Jeffreys minisatellite DNA. The use of this new sample for gene fingerprinting has shown its high efficiency and suitability for solving many problems. The fact is that these hypervariable sequences are found in various representatives of living nature - humans, animals, plants and bacteria, and therefore the M13 bacteriophage DNA probe can be used on a wide scale. For example, for personal identification, to establish the kinship of any living beings. The method makes it possible to solve problems of genetics and selection of animals, to select for useful traits; Using this method, it is possible to carry out genetic certification of individual highly productive animals, analyze the pedigree and use the obtained information for targeted selection.

There is another type of DNA hybridization analysis - this is the point (dot) hybridization method (Fig. 9), which is performed by introducing the DNA samples under study in a denatured state onto nylon membrane filters in the form of dots. For example (Fig. 12.5), the DNA of Mycobacterium tuberculosis large cattle in an amount of 3 μl (1.8 μg/ml) in the form of dots is applied to squares (1.5 x 1.5 cm).

Rice. 12.5. Dot hybridization of M.bovis DNA probe: A: 1 - M.bovis: 2.3, - DNA from tuberculosis-affected tissue; B: 1,2,3 - DNA isolated from tissues of healthy animals; C: 1 - DNA of the causative agent of brucellosis;

2 - DNA of the pathogen Listeria;

3 - M. fortuitum DNA.

Single-stranded DNA molecules (denatured) are adsorbed on the membrane and fixed. After this, a DNA probe labeled with radioactive phosphorus, that is, a single-stranded M. bovis molecule labeled P 32, is applied to the filter. Since in this case the nitrogenous bases of the M.bovis DNA molecules and the P 32 labeled DNA probe are complementary, the nitrogenous bases of the DNA strands and the DNA probe bind to form double helix. After this, unbound DNA probe molecules are washed off and the resulting hybrid molecules are detected by autoradiography.

Once DNA, RNA or proteins are separated, they must be transferred to a solid support for detection and other operations that are difficult to do in a gel. The transfer process leading to immobilization of molecules , i.e. fixed in a stationary state is called blotting (in English. - blotting ). Nylon or nitrocellulose membranes are used as a substrate.

Blotting(from the English blotting - blotting) is a method of transferring electrophoretic DNA fragments onto a special film (membrane) made of nitrocellulose that binds (immobilizes) single-stranded DNA molecules.

Southern blotting(after the name of the author who proposed it) is based on the movement of DNA fragments due to the capillary effect. The process of transferring DNA fragments contained in an agarose gel onto a nitrocellulose film using filter paper is similar to blotting.

The analysis is carried out as follows:

– Isolated, purified, denatured and fragmented DNA is placed on a sheet of agarose gel, where fragments are electrophoretically separated by mass and charge.

– A sheet of agarose gel is placed on filter paper moistened with concentrated saline (buffer) solution.

– Then a nitrocellulose filter is applied to the gel, where immobilization (or adsorption, or fixation) of single-stranded DNA fragments occurs.

– A stack of sheets of dry filter paper is placed on top of the filter, which ensures a slow flow of the buffer solution through the gel (i.e., serves as a kind of capillary pump). The saline solution, passing through the agarose gel, carries with it DNA fragments, which are retained and bound by nitrocellulose, and the solution is absorbed by dry filter paper.

– Next, the DNA is denatured with alkali, and the filter is kept in a vacuum at a temperature of 80 0 C, as a result of which single-stranded DNA fragments are irreversibly immobilized (fixed) on nitrocellulose. In this case, the location of the immobilized DNA bands exactly corresponds to their location in the gel.

– DNA bound to the filter is placed in a solution with a DNA-labeled probe, in which hybridization occurs. Only DNA fragments complementary to it will hybridize (form hydrogen bonds) with a specific probe, which can be detected as light stripes on X-ray film, i.e. autoradiography of a nitrocellulose filter

Dot blot. To prepare dot blots, a DNA or RNA preparation is applied directly to the filter. Droplets of the drug look like dots on the filter, which explains the name of the type of blotting (English: dot). 1) From genomic DNA pre-treated with ultrasound, fragments 5–10 nucleotide pairs long are formed.

2) To make DNA or RNA probes accessible to the probe, they need to be denatured, i.e. convert to single-chain form. This occurs under the influence of a temperature of 100 °C.

3) Denatured nucleic acids are incubated on ice: a rapid decrease in temperature prevents their renaturation, i.e. complementary pairing of chains. Denatured DNA or RNA is applied directly to the filter, which is incubated in a solution containing the probe.

4) To prevent the analyzed nucleic acid from going into solution, it must be fixed on a filter (membrane). For this, two types of filters are used: nitrocellulose and nylon.

To immobilize nucleic acids on a nitrocellulose filter, frying is used at 80 °C in a vacuum, and on a nylon filter, UV irradiation is used for 3–5 minutes.

5) After incubation of the nucleic acid preparation with an isotope-labeled probe, autoradiography is carried out in a special cassette or identification using non-radioactive methods.

Dot blotting allows you to answer only one question: whether the desired nucleotide sequence is present in a given sample.

Northern blot analysis applies:

1) to isolate and analyze RNA (for example, to determine whether mRNA read from a given gene is present in a given cell type, i.e. whether the gene is expressed or not;

2) to determine the amount of this RNA and its changes in the development of a given cell type;

3) to determine the size of a transcript of a gene.

In this case, RNA molecules isolated from the cell are separated by size using gel electrophoresis and then transferred to a filter. After hybridization with a labeled single-stranded probe, the sites of hybridization (homology) of the RNA and the probe are identified.

If the nucleotide sequence of the desired gene (or mRNA) is not known, but the protein whose synthesis it controls is known, then it is possible to isolate a small amount of pure protein and determine the amino acid sequence of some of it (knowledge of 5–6 amino acid residues is sufficient). Using the table genetic code, it is possible to establish all possible nucleotide sequences in the section of mRNA (or the gene itself) that encodes a given amino acid sequence. In this case, a probe can be synthesized to search for the desired clones in a gene library.

Western blottinG(immunoelectroblotting, protein blotting) is a method for identifying unique proteins. It is based on the phenomenon of highly specific antigen–antibody interaction. Thus, the antigen (target) is the protein being determined, and the probe is the antibody to it.

Antibodies to the protein under study are obtained different ways. The simplest is to inject a purified protein sample into the bloodstream of a laboratory animal (usually a rabbit). His body produces antibodies (immunoglobulins) to this foreign protein. These are primary antibodies that will interact with the target protein.

However, it would not be rational to introduce an identification label directly into the antibody data. Detection of different proteins would require labeling different antibodies, which would result in high costs. It turned out to be more reasonable to use universal antibodies – conjugated antiimmunoglobulins, which are essentially antibodies to antibodies produced by using the identified protein as an antigen. For example, conjugated anti-immunoglobulins to rabbit Ig will interact with all immunoglobulins synthesized in rabbits to different antigens. Thus, it is precisely such universal secondary antibodies that carry an isotopic or non-radioactive label. In addition to a non-isotopic label, which in the course of a number of reactions leads to the formation of an insoluble colored compound (as in the case of nucleic acid blotting), a chemiluminescent label, which has a higher sensitivity, is very often used.

1) Extraction of proteins from homogenate

2) Separation of proteins by molecular weight using SDS-polyacrylamide gel electrophoresis (PAGE). The SDS electrophoresis method involves denaturation of native proteins. Thus, protein molecules that have the same molecular weight, will travel the same path in the gel and line up in the form of a strip. Since protein molecules of different sizes are present in the mixture, many bands are formed. Electrophoresis results can be visualized by protein staining (Coomassie brilliant blue, amido black, silver staining). Silver staining has a unique sensitivity that allows detection of as little as 0.1 ng of protein in the resulting band. This is very important to control the amount of protein applied to the gel.

3) Transfer of proteins from the gel to the membrane. This is done because polyacrylamide does not allow large immunoglobulin molecules to diffuse into the protein. And the protein immobilized on the membrane becomes accessible to antibodies. Unlike nucleic acid blotting, protein transfer to the membrane occurs under the influence of electrical forces, i.e. in an electric field.

4) The resulting blot is incubated with antiserum to the protein, and then with antiimmunoglobulins. The result is visualized according to the label type used.

Restrictions:

1) the large size of the fragments under study, significantly exceeding the length of DNA probes and preventing direct molecular analysis;

2) the impossibility of arbitrarily choosing the ends of the studied sequences, determined by the presence of corresponding restriction sites in the original DNA molecule;

3) necessity large quantity well-purified high molecular weight genomic DNA (at least 10 μg per reaction, which is equivalent to 0.5-1 ml of blood),

4) for genomic hybridization - the presence of radioactive DNA probes with high specific activity of at least 109 pulses/min * µg), operating for a limited period of time, and a specially equipped isotope unit. In addition, prolonged exposure of autographs significantly lengthens the time to obtain results.

5) high labor intensity research

Blotting (literally - blotting) is the transfer of fragments of macromolecules (DNA, RNA or protein), separated by gel electrophoresis, onto a solid support - a membrane. In studies of the human genome, the blotting method developed by Southern is often used, in which an oligonucleotide probe in solution is hybridized with DNA adsorbed on a membrane (Southern blot hybridization). Genomic DNA (usually isolated from leukocytes or fetal cells) is cleaved into short fragments, separated in an agarose gel, transferred to a membrane, and then specific regions are identified using hybridization with oligonucleotide probes (Fig. 65.6). This method identifies unique DNA fragments, the size of which is approximately one millionth of the genome.

The significance of the method for medicine is due to the ability to study a certain fragment of genomic DNA in any person.

The method is used to detect large rearrangements in DNA and some point mutations (most point mutations cannot be detected by this method).

Denaturation of DNA is carried out with alkali. After electrophoresis is completed, the gel is placed in a base (alkali) solution, in which double-stranded DNA fragments lose their bonds and become single-stranded.

Transfer of DNA from the gel to a nitrocellulose or nylon filter is carried out in a buffer solution. A filter and a stack of filter paper are placed directly on the surface of the gel. Due to the capillary effect, a buffer current is created perpendicular to the gel plane. The DNA washed out of the gel is retained by the filter and almost completely ends up on its surface. After transfer, the single-stranded threads are fixed on the filter. The location of the fragments on the filter exactly matches their location in the gel. 3. In order to visually identify the required fragments (DNA fixed on the filter is not visible), hybridization is carried out with a specific nucleotide sequence labeled with radionuclides or a fluorescent label, an oligonucleotide synthetic probe (such a probe consists of 16-30 base pairs), or a cloned DNA fragment. The nucleotide sequence of the probe must be completely or partially complementary to the region of genomic DNA being studied.

When the filter is incubated with a solution containing a labeled probe, hybridization of the complementary DNA strands of the probe and the fragment on the filter occurs. Nonspecific bound probe molecules are washed away using a special procedure.

Radioactively labeled areas are detected by exposing a filter to X-ray film (autoradiography). After development, bands of probe-labeled DNA are visible on the film.

Non-radioactive labels are visualized by fluorescence or indirectly by antibodies.

So: Southern blot hybridization is a method for analyzing the structure of a given genome region, based on:

2) separating the plurality of resulting fragments using electrophoresis in a flat plate of agarose or other gel;

3) transferring the separation products onto a sheet of porous material, for example, onto a nitrocellulose filter in such a way that all DNA fragments are transferred to the filter and fixed on it, creating an imprint identical to the distribution of fragments in the gel after separation;

4) hybridization of the filter with a radioactively or dye-labeled fragment of DNA or RNA (probe), which in sequence corresponds to the analyzed region of the genome. As a result, the sample, due to complementary interactions, binds to those parts of the filter where the genome fragments under study are located, and the position of these fragments is identified by the position of the tag from the sample on the filter.

And as mentioned above, with the help of Southern blot hybridization it is possible to determine the identity or difference in fragment lengths (restriction fragment length polymorphism, RFLP) obtained by restriction cleavage of the same locus in different compared genomes.

Peculiarities. Southern blotting in molecular genetics. Features In vivo DNA cloning

Method

results

Application

Notes

see also

See what “Southern blotting” is in other dictionaries: