Hybridization of nucleic acids. Southern and northern blotting

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 from 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.

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Notes

Excerpt characterizing Southern blot

– Immortality, dear... Just immortality. But, unfortunately, he does not understand that it is not given simply because someone wants it. It is given when a person is worth it, when he KNOWS what is not given to others, and uses it for the benefit of other, worthy people... When the Earth becomes better because this person lives on it.
- Why does he need it, mom? After all, immortality is when a person must live for a very long time? And this is very difficult, isn’t it? Even for my own short life everyone makes many mistakes, which he then tries to atone for or correct, but cannot... Why does he think that he should be allowed to make even more of them?..
Anna shocked me!.. When did my little daughter learn to think completely like an adult?.. True, life was not too merciful or soft with her, but, nevertheless, Anna grew up very quickly, which made me happy and alarmed at the same time ... I was glad that every day she was becoming stronger, and at the same time I was afraid that very soon she would become too independent and independent. And it will be very difficult for me, if necessary, to convince her of something. She always took her “responsibilities” as a Sage very seriously, loving life and people with all her heart, and feeling very proud that one day she could help them become happier, and their souls cleaner and more beautiful.
And now Anna met for the first time with real Evil... Which mercilessly burst into her very fragile life, destroying her beloved father, taking me, and threatening to become a horror for herself... And I wasn’t sure if she had enough strength to fight everything alone in case her entire family dies at the hands of Caraffa?..
The hour allotted to us passed too quickly. Caraffa stood on the threshold, smiling...
I'm in last time I hugged my beloved girl to my chest, knowing that I wouldn’t see her for a very long time, and maybe even never... Anna was leaving for the unknown, and I could only hope that Caraffa really wanted to teach her for his crazy people goals and in this case, at least for some time nothing threatens her. For now she will be in Meteora.
– Did you enjoy the conversation, Madonna? – Caraffa asked feigningly sincerely.
– Thank you, Your Holiness. Yes, of course. Although, I would prefer to raise my daughter myself, as is customary in the normal world, and not give her into the hands of unknown people, just because you have some kind of plan for her. There isn't enough pain for one family, don't you think?
- Well, it depends on which one, Isidora! – Karaffa smiled. – Again, there is “family” and FAMILY... And yours, unfortunately, belongs to the second category... You are too strong and valuable to just live like that without paying for your opportunities. Remember, my “great Witch,” everything in this life has its price, and you have to pay for everything, regardless of whether you like it or not... And, unfortunately, you will have to pay very dearly. But let's not talk about bad things today! You had a wonderful time, didn't you? See you later, Madonna. I promise you, it will be very soon.
I froze... How familiar these words were to me!.. This bitter truth accompanied me so often in my still short life that I could not believe that I was hearing them from someone else!.. This is probably what It was indeed true that everyone had to pay, but not everyone did it voluntarily... And sometimes this payment was too expensive...
Stella peered into my face in surprise, apparently noticing my strange confusion. But I immediately showed her that “everything is fine, everything is fine,” and Isidora, who fell silent for a moment, continued her interrupted story.
Caraffa left, taking my dear baby away. The world around us faded, and my devastated heart, drop by drop, was slowly filled with black, hopeless melancholy. The future seemed ominous. There was no hope in him, there was no usual confidence that, no matter how difficult it was now, in the end everything would work out somehow, and everything would definitely be fine.

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Southern blotting

Southern blotting 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 onto a membrane filter for hybridization. The method is named after its inventor, English biologist Edwin Southern.

Other transfer 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, the gel is processed before transfer, e.g. hydrochloric acid, which causes depurination of DNA and facilitates transfer to the membrane.
When using the alkaline transfer method, the agarose gel is placed in alkaline solution, while the DNA double helix denatures and facilitates the binding of negatively charged DNA to a positively charged membrane for further hybridization. At the same time, the remaining RNA is also destroyed.
A sheet of nitrocellulose 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. 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.
Hybridization of a radioactively labeled sample with a known DNA sequence is carried out with a membrane.
After hybridization, excess sample is washed from the membrane and the hybridization products are visualized by autoradiography or the color of the membrane is assessed.

Reporter gene

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 target mRNA is less than 1 in 1000 total number 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 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 areas 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-stranded labeled probe under conditions that promote the pairing of double DNA molecules. Unbound probes are removed by washing, then the filter with the 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.

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). Saline solution, passing through an agarose gel, carries with it DNA fragments that are retained by nitrocellulose and bind to it, 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. Hybridize (form hydrogen bonds) with a specific probe there will only be DNA fragments complementary to it, which can be detected in the form of light stripes on X-ray film, i.e. autoradiography of a nitrocellulose filter

Dot blotting. 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.

For immobilization 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 in various 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. Identifying different proteins would require different antibodies to be labeled, resulting in different high cost. 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) the need for a large amount of 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