Northern blotting technique

Electrophoretically separated RNA molecules get transferred from the gel to the absorbent sheet, immersed in a labeled probe for hybridization. Thus, the northern blotting follows the same steps of Southern blotting. However, RNA instead of DNA gets blotted. The standard Northern blotting procedures help to compare the quantities of the transcripts from different tissues. The technique’s sensitivity increases to a 100 fold using an mRNA or a messenger RNA. The technique analyses many types of RNAs such as micro RNA, small nuclear RNAs or snRNAs, small interfering RNAs or siRNAs, and mRNA. The technique is helpful in RNA interference studies or RNAi technology. It also involves studying the expression of oncogenes. Northern blotting includes human RNAs, plant, and animal RNAs. The technique has placed itself very well in genetic research and engineering.

Image: Northern blotting

The description of the blotting procedure is as follows:

Step 1: Isolation of mRNAs:

RNA extraction uses a homogenized tissue sample. Oligo (dT) cellulose chromatography helps in isolating mRNA with a poly (A) tail. We know the mechanism of polyadenylation well. It protects the RNA from degradation by adding polyadenine nucleotides to the RNA molecule. Widely used methods include phenol-chloroform extraction or trizol method.

Step 2: Electrophoresis:

Once the RNA isolation gets completed, the loading of the samples in the wells helps to obtain the bands. Separation of the RNA molecules involves agarose gels most of the times. Fragmented RNA or micro RNA separation may include polyacrylamide gels. Formaldehyde acts as a denaturing agent in the electrophoresis. It limits the RNA secondary structure. Fragment sizes comparison uses a ladder RNA in another well. Electrophoresis involves staining the gel with ethidium bromide. Handling ethidium bromide requires caution because of its carcinogenicity and neurotoxicity. Ethidium bromide intercalates the RNA, thereby acting as an intercalating agent.

Step 3: Using a nylon membrane:

After separation of RNA molecules through gel electrophoresis, the samples get transferred to a nylon membrane through a capillary system. Following is the apparatus for northern blotting:

The first step involves filling a tray with a buffer solution consisting of formamide. It helps in lowering the annealing temperature of the probe-RNA interaction thereby preventing RNA from getting degraded. Then the RNA gets immobilized to the membrane through a covalent linkage. UV light or heat facilitates this property. The blotting paper used in this apparatus plays a role in capillary action by carrying the buffer solution through the gel. Once the RNA interacts with the buffer, it gets transferred to the membrane. A stack of paper towels and weight kept on the membrane enhances the imprint of the bands on the membrane. Now the RNA molecules present on the membrane exactly resemble as they were on the gel.

Step 4: Hybridization with the labeled probe:

Exposure of the membrane to the probe ensures hybridization of RNA molecules. Northern blotting procedure involves probes composed of complementary sequences of RNA. It consists of at least 25 complementary bases. Northern blotting uses cDNA as a probe. Radioactive labeling of the probe includes a radioactive isotope P³². Alternative labeling involves non-radioactive techniques such as chemiluminescence labeling. The chemiluminescence technology involves the breakdown of the chemiluminescence substrates through enzymes such as alkaline phosphatase or horseradish peroxidase. The substrates produce a detectable emission of light.

Step 5: The procedure of chemiluminescent labeling:

The probe (cDNA) gets attached to the enzyme alkaline phosphatase or horseradish peroxidase. Alternatively, labeling of the probe may involve a ligand. The ligand gets attached to the enzyme. Determination of the efficiency of the hybridization includes ionic strength, viscosity, duplex length, and base composition. Non-hybridized probes get removed by washing the membrane filter gently.

Step 6: Exposure to the X-ray film:

An X-ray film detects the signals generated with the chemiluminescent-labeled probes hybridized to RNA. A quick and sensitive signal generation occurs through chemiluminescent labels.

Step 7: Quantification of RNA through densitometry:

The process of densitometry involves a quantitative measurement of the optical density in the light-sensitive material.

Following is the summary of the northern blotting procedure:

1.     Isolation of RNA from a tissue sample.
2.     Loading the samples and the markers in the electrophoretic wells.

3.     Fragments get separated based on their sizes. Molecules with larger sizes are near the wells. Molecules with smaller sizes move faster. The fragments appear in the form of bands.

4.     Exposure of the gel to a buffer solution and a membrane filter.

5.     RNA gets transferred from the gel to the membrane.

6.     RNA hybridization using probes.

7.     Signal detection

8.     RNA quantification

Applications of northern blotting:

Gene expression study includes the pattern of gene expression in the tissues, organs and developmental stages. Northern blotting applies in studying the overexpression of oncogenes, the upregulation or downregulation of oncogenes and tumor-suppressor genes. It may help to find a gene function. Northern blotting helps to check the cloned DNA. Especially it checks the cloned cDNA since the technique uses cDNA as a probe to detect specific RNA. Analysis of micro RNAs becomes easy with this technique. A high resolution northern blotting monitors RNA expression. Micro RNAs are short and non-coding regulatory molecules. Post-transcriptional regulation of genes involves the role of micro RNA. The technique is readily available. Hence it is used in micro RNA analysis. The probes used in the technique are known as locked nucleic acid modified oligonucleotide probes. They are extremely sensitive and specific in detecting mature micro RNAs.

Immuno-northern blotting detects RNA modification through antibodies. The RNA gets separated through electrophoresis and transferred to the membrane. Immuno-blotting involves antibodies. It reveals antibody cross-reactions, characterization of antibodies, and modified nucleosides. It is a highly specific technique. The northern blotting technique also characterizes the RNA interference reagents. The comparison of the data derived from deep sequencing of micro RNAs with endogenous and exogenous RNAs is possible with the northern blotting technique. A gene silencing phenomenon is known as RNAi or RNA interference. The double-stranded RNAs get processed into small interfering RNAs (siRNA). A siRNA acts like a guide and enables cleavage of a homologous RNA. It occurs mainly in the RNA induced silencing complex (RISK). High-resolution northern blotting efficiently detects the length heterogeneity of the RNAi technology reagents.

A blot-base is an online database used to publish northern blots. It is a database used in genome sequencing, determination, and the protein structure.

Reverse northern blotting:

It is a variant of northern blotting technique. In this, the DNA fragments get hybridized with the RNA probes labeled radioactively. It enables gene expression profiling.

Virtual northern blotting:

It involves a comparison of relative amounts of transcripts in different tissues. The comparison is between small quantities of total RNA and full-length cDNA.

Advantages and disadvantages of northern blotting:

Detection of RNA size and observation of alternate spliced products become easy with this method. The technique is applicable for quantitative as well as qualitative analysis. It has a high specificity. With so many advantages, there are disadvantages too. As compared with RT-PCR, northern blotting has a low sensitivity. Analyzing thousands of genes at a time is not possible. The RNases are always ready to degrade the sample. Hence the technique always requires RNase inhibitors and proper sterilization of glassware. Chemicals used in northern blotting may be risky.

References:
[1] Molecular Biology Techniques: An Intensive Laboratory Course, Walt Ream, Katharine G. Field

[2] Ana Techniques in Biotechnology, Goutam Bhowmik

[3] Gene Cloning and DNA Analysis, T.A. Brown
[4] Molecular Plant Biology: A Practical Approach, Volume 2, Philip M. Gilmartin, P. M. Gilmartin, Dr. Chris Bowler

                                  © Copyright, 2018 All Rights Reserved.

A Review on Gene Cloning

Suppose you have generated important data and you do not want to lose it. What would you do to protect it? You may either file it or store it in a folder. Or you would generate multiple copies of the information using a Xerox. The Xerox machine reads the information in your paper and gives you a printout in the form of a photocopy. Thus, Xerox creates exact replicas of the document. Similarly, our body protects the genetic material by generating multiple copies. An example involves the replication process.
Scientists felt the need to generate multiple DNA copies. The process of cloning involves obtaining multiple copies of a gene or a DNA segment using technology. Each identical copy of a DNA segment or a gene segment is known as a clone. Human genome project utilizes maximum amounts of clones in various experiments. Many such cloned DNA collections make up a clone library. Cloning is not only applicable to DNA or a gene segment but also possible with the entire cell. This type of cloning exploits the natural process of cell division to make many copies of an entire cell. The genetic makeup of the cloned cells is known as cell lines. They are identical to the original cell. Gene cloning involves recombining a population of DNA molecules into the vector. The recombined DNA molecules contain the gene of interest. The DNA is inserted into a vector so that each vector consists of single DNA from the original population. The screening of the colonies revealed the presence or absence of the DNA of interest. Thus, understanding cloning requires knowledge of the clones and their production.

Image 1: Gene Cloning

The review article discusses various aspects of cloning:

1.     Restriction enzymes and cloning vectors

2.     Collection of clones in the form of a library

3.     Screening of specific clones

4.     Analyzing cloned DNA through a molecular approach

5.     Identifying adjacent clones

6.     Applications

Cloning involves producing multiple copies of the genes or DNA segment of interest. In short, the procedure involves digesting a DNA segment with restriction enzymes, introducing it in a vector, transforming the recombinant vector into the host and observing the expression of the gene of interest. The screened clones are later on analyzed using molecular techniques.

Restriction digestion:

The restriction enzymes are used to digest the DNA. These enzymes are endonucleases capable of cleaving specific sites in the DNA known as the restriction sites. The restriction sites have specific nucleotide base pairs. A restriction enzyme cleaves a specific base by breaking the phosphodiester bond. Cleaving of the DNA can be achieved using one or a combination of restriction enzymes. The process of cleaving the DNA to obtain fragments of different sizes is known as restriction digestion. Separation of the DNA fragments is possible with the electrophoretic technique. Smaller fragments travel faster than the larger fragments, thereby forming bands. After visualizing bands, the portion of the gel consisting of a DNA fragment is cut and used for cloning experiments. Here are the two options. One of the options is to make multiple copies of the gene using amplification techniques such as PCR. The second option is to incorporate the DNA into the cloning vector. PCR or polymerase chain reaction is a process of amplifying the gene or a DNA using a sophisticated thermal cycler. Cloning DNA using a vector involves inserting the desired DNA into a plasmid or another vector.

Cloning vectors:

A cloning vector replicates within the host organism. It consists of a restriction site which can be cleaved for inserting the desired DNA fragment. Various cloning vectors are available. A plasmid vector is a plasmid or an extrachromosomal material of the bacteria. It is isolated and used as a vector. It consists of three sites such as the origin of replication, a selectable marker gene, and one or more restriction sites. For example, pUC 19 vector has a polylinker site or multiple cloning sites. On treating the restriction site with a restriction enzyme, that particular site gets cleaved and the desired fragment of DNA gets inserted in that place. Other examples of vectors include shuttle vectors, expression vectors, cosmids, phasmids, and artificial chromosomes. Shuttle vectors are bifunctional. They are capable of insertion into two or more hosts. Expression vectors express the desired gene to get the protein product.

Clone Library:

It can be anything from a collection of clones including cDNAs, chromosomes, and genomes. A cDNA library is nothing but a collection of cloned cDNA sequences synthesized from mRNA. A collection of cloned DNA in which the sequence appears at least once is known as a genomic library. The clone libraries appear analogous to book library. Just as the books provide plenty of information, the clone libraries provide a lot of information about the genome. Genome libraries production involves inserting the required DNA digest into a vector and storing the product. However, there are other methods too. Gene splitting with a restriction enzyme sometimes gives the undesired product. Such cases involve other techniques like mechanical shearing or partial digestion. Large DNA insertion involves mechanical shearing. The process of screening is made easy using chromosome libraries. 24 different human chromosomes libraries are available. The preparation of the cDNA libraries involves mRNA molecules as raw materials. After making clone libraries, a specific gene is found using screening techniques.

Image 2: Transformation of the recombinant plasmid

Screening procedures:

The screening of cDNA libraries involves an antibody probe. The first step is to transform the recombinant plasmid consisting of the required cloned cDNA into a host such as E. coli bacteria. Next step involves plating the bacteria on a selective medium to observe the growth of the colonies. Transferring the colonies into a microtiter well enables the bacteria to grow. The transfer of the colonies to the membrane filter also enables them to grow to get the expression of the gene. Removal of the filter and cell lysis enables the protein product exposure. Radioactively labeled antibody treatment with the protein product is the final step. Autoradiography helps the observation of clones in the form of dark spots. Screening a genomic library involves a similar system. It involves plating the transformed bacteria on a medium and processing them through replica plating. Here, the probed DNA is used instead of a radioactively labeled antibody. Complementation test helps to identify the specific genes.

Analyzing cloned DNA:

Analysis of clones involves molecular biology techniques such as restriction mapping, southern and northern blotting. Restriction mapping includes the physical mapping of the genes. Restriction digestion gives rise to DNA fragments of different sizes. Electrophoresis separates the fragments in the form of bands. Southern blotting uses a nitrocellulose membrane filter placed on the gel immersed in the buffer solution. Capillary action of the blotting paper enables the DNA to come in contact with the buffer and transfer to the membrane filter. The filter gets exposed to probes which hybridize with the DNA. Autoradiography helps to detect the DNA. Southern blotting mainly involves blotting DNA. Northern blotting involves blotting RNA instead of DNA.

Adjacent clone identification:

Genes between the flanking markers are found through chromosome walking. Hence, adjacent clone identification requires chromosome walking. The overlapping clones help to study larger DNA segments.

Applications of cloning:

1.     Recombinant DNA technology or genetic engineering: Cloning techniques involve obtaining large amounts of pure DNA. Expression of the desired products including proteins, vitamins, enzymes, growth factors, and other biomolecules is possible with recombinant DNA technology. Cloned genes help to detect mutations. Plant vectors consisting of the desired gene improve the crop quality. Cloning enables development of transgenic plant varieties with stress tolerance, drought tolerance, and herbicide or pesticide resistance. Particular protein synthesis is achieved using expression vectors.

2.     Transgenics: These organisms, plants, animals or microbes, involve manipulated genes cloned into several copies. The genes of interest express phenotypically. Examples include knockout animals.

3.     Gene therapy: It helps in replacing the gene that has lost its function. The defective gene identification accompanies cloning the normal gene inserted into the affected individual through a vector. The first gene therapy was successful in children suffering from ADA deficiency. The gene therapy is a new way to treat genetic disease because the abnormal gene is corrected or replaced.

4.     In vitro fertilization: Microsurgery involves removal of the nucleus from the donor’s oocyte. The microinjection of the surrogate mother’s somatic cell nucleus into the donor’s oocyte makes the cell chimeric. The chimeric cell cultured in the laboratory develops into an embryo. The embryo gets implanted into the surrogate mother’s uterus.

     References:
[1] Gene cloning and DNA analysis, T.A. Brown
[2] Recombinant DNA Technology, Sardul Singh Sandhu
[3]Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh
[4] Gene Cloning and Manipulation, Christopher Howe

   

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