Gene cloning or creating multiple copies of the desired gene and other sequences gets analyzed using various techniques. Cloning studies require information related to genes such as the position or the location of a gene on a chromosome, specific restriction sites, and their special arrangement. Cleaving the DNA with specific restriction enzymes help to target specific regions known as the restriction sites. A plethora of gene analyzing techniques involves restriction mapping, Southern blotting, northern blotting, and many others. Plasmid genomic libraries for specific DNA sequences or cloned DNA sequences get analyzed using DNA probes capable of hybridization. Labeling of the probes may or may not require radioactive labels. Techniques such as Southern or Northern blotting utilize probes. The probes have important properties. They help in recognizing complementary sequences in the nucleic acid molecule. It becomes simpler to identify and isolate specific DNA sequences from an organism. They play an important role in diagnostics and fingerprinting techniques.

The difference between the southern and northern blotting is simple. The Southern blot helps to blot the DNA. Northern blot helps in blotting RNA. Both the techniques involve electrophoresis for separating the nucleic acid fragments and probes for hybridization techniques. It is not possible to separate the DNA into fragments without digestion. The separation of fragments using electrophoresis requires a properly digested DNA. Hence, restriction mapping and blotting techniques go hand in hand.





Image: Cloned gene analysis





Restriction mapping:

A restriction mapping technique typically uses certain enzymes possessing the ability to cleave the DNA into fragments of particular sizes. The necessity of a restriction map is to locate the genes present on the chromosomes, study mutations associated with each polymorphism and lot more. Thus, restriction maps act like guides in cloning and molecular biology studies. A restriction enzyme cleaves the DNA at a particular site known as a restriction site. A nucleotide sequence specific to an endonuclease enzyme is known as a restriction site. For example, Eco RI, a restriction endonuclease, cleaves at guanine base of a 5’-GGATTC-3’ recognition sequence. The process of treating the DNA with the restriction enzyme is known as restriction digestion. The products obtained from the restriction digestion are known as restriction digests. A restriction digest consists of fragments of DNA of different sizes. In most of the restriction digestion experiments, the DNA sample gets treated with one or more restriction enzymes. The DNA samples get separately labeled. If one DNA sample gets treated with a particular restriction enzyme, the fragments obtained through this process get separated using electrophoresis. Suppose a DNA sample gets treatment with restriction enzyme I whereas the other one gets treated with restriction enzyme II.

Consider one more DNA sample treated with a combination of restriction enzyme I and II. After fragmenting the DNA samples, the next step involves loading the samples into the electrophoretic wells. Cleaving the DNA fragments with a single restriction enzyme reveals two bands. Cleaving a DNA sample with two restriction enzymes reveals three bands on the electrophoretic gel. The DNA fragments get separated based on their sizes. The smaller DNA fragments migrate faster. The larger DNA molecules migrate slowly. The comparison of the bands involves a marker DNA and control samples. The sizes of the fragments are noted down. A calibration curve construction involves distance migration on X-axis and Log Kb on the Y-axis. The interpretation of the results requires construct models. Restriction mapping is a kind of physical mapping technique. It helps in sequencing and analyzing the cloned DNA fragments. It is possible to find out whether a vector gets cloned properly using restriction mapping. Restriction digests get electrophoresed further for Southern blotting.





Southern blotting:

The electrophoresed gel consisting of DNA fragments get separated as per the sizes. Then it gets analyzed using hybridization and blotting procedures. The Southern blotting technique helps in blotting DNA bands. Discovered by E.M. Southern, this technique has a wide range of applications in cloning, SNP analysis, molecular testing, RFLPs, zoo blots, DNA fingerprinting, and microarray studies.

When DNA fragments get treated with the restriction enzymes and separated by gel electrophoresis, the bands of different sizes get separated on the gel. The next step involves placing the gel in a tray full of alkaline solution (buffer solution). The gel immersed in a buffer solution comes in contact with the buffer. The technique also utilizes a glass plate or a blotting paper. Next step involves covering the gel with a nitrocellulose filter paper. A stack of paper towels and weight gets placed on top of it. The blotting paper acts as a wick and carries the buffer solution from the tray to the gel, and finally to the membrane filter. The DNA fragments present on the gel come in contact with the buffer and get transferred to the membrane filter. Now, the membrane filter looks like a replica of the gel with distinct bands. The membrane gets further treated with the probe solution so that the hybridization takes place. Later on, the technique known as autoradiography helps in visualizing the bands. The Southern blotting technique helps to analyze cellular DNA for the presence of sequences complementary to the labeled probes. The cDNA molecule gets synthesized from a mRNA molecule and analyzed using Southern blotting.





Northern blotting:

It is a technique used to study RNA rather than DNA. It is similar to the Southern blotting technique. This technique reveals the size of the mRNA. It is used to investigate the presence of mRNA in a particular cell type or a tissue. The levels of gene activity get determined during the developmental stages. Once the electrophoretically separated RNA passes from the gel to an absorbent sheet, the RNA of interest gets revealed after hybridization. Transcription fo specific gene under certain environmental conditions gets detected using Northern blotting. Unregulation and downregulation of oncogenes and tumor suppressor genes can be studied.