Screening of clones

Just like collecting books and arranging them in a library, the recombinant DNA library stores the cloned DNA fragments. For selecting a particular clone, we need to screen these libraries. Hence, before knowing the screening procedures, we must know the types of libraries. There are two main types of libraries such as cDNA library and genomic library. The cDNA library involves a collection of cloned cDNAs produced from a mRNA. The DNA sequence gets represented at least once in a genomic library.

1.   Screening a cDNA library:

For screening, we need to search for a cDNA clone encoding a protein. An antibody probe screens specific cDNA plasmid. It involves the use of an expression vector. Since we need to search for a cDNA clone encoding a protein, we use expression vectors. An expression vector is nothing but the vectors designed for the expression of the genes. When the cDNA gets cloned into an expression vector, the gene gets expressed and produces proteins. The cDNA gets inserted into an expression vector such that it gets fixed between the promoter and the termination signal. When cDNA gets inserted in the host, the gene gets expressed. The selection of the host involves E. coli cells. The expression vector containing cDNA gets inserted into the bacteria through the process of transformation. Why only expression vectors? An expression vector expresses the genes efficiently. Screening the cDNA clones requires a specific host such as E. coli. The growth of the cells helps us in identifying the recombinants and non-recombinants. It depends on the expression of the cDNA or the desired gene. Designing an expression vector depends on the type of organisms such as a prokaryote or a eukaryote. Hence, for every type of an organism, an expression vector design gets ready. Plus, we add a purification tag or a fusion partner along with the cDNA or a cloned gene. It helps in obtaining a purified product.

Plating the bacterial cells (transformed with the vector) on a selective medium allow the bacterial colonies to grow. Individual colonies get picked up and transferred to a microtiter dish. It is a 96 well dish. A microtiter well serves as a liquid holder. Several milliliters of liquid gets filled in the microtitre dish or well. Microtitre dish or a well has many applications. They serve in screening, filtration, separation, storage, culturing cells, detecting the anti-microbial activity, and many other uses. Microtiter dishes adjust with the temperatures. The evaporation of the solvent occurs more after heating.

Next step involves a pertiplate with a selective medium. A membrane filter placed on the Petri plate for getting the replicas of the clones. The selective medium is appropriate for the recombinant molecules. The pattern of the growth of colonies on the membrane filter is the same as the colonies growing on the microtiter wells. After observing or allowing growth of the colonies, the filter gets peeled from the dish. The cells get lysed using a lysis solution in situ. The protein product of cDNA gets bound to the filter. Next step involves incubation with an antibody labeled radioactively. The placement of the filter in the X-ray film helps in observing the expressed protein of interest. This process is also known as autoradiography. The development of X-ray film gives dark spots in the region where radioactive probe gets bound.

Image 1: Procedure for Screening of clones

Image 2: Antibody-Protein interaction

2.   Screening a genomic library:

The cloned gene coding for mRNA molecule gets identified using plasmid genomic libraries. The technique involves DNA probes. The construction of the genomic libraries depends on a vector such as a plasmid vector. The procedure for screening a genomic library is similar to that of the cDNA library. The host used in screening procedures involve E. coli bacterial cells. The first step involves the transformation of genomic libraries into E. coli cells. The cells plated on a selective medium help in obtaining the growth of the colonies. The technique involves replica plating. It helps in producing identical copies of a series of the bacterial colonies on Petri dishes. First, the Petri plate consisting of the bacterial colonies gets inverted. The surface of this dish gets pressed against a cylindrical block and with a velveteen cover. Hence, the bacterial colonies present on the Petri-plate get transferred to the velveteen. There is an extension to this procedure. The pressing of the velveteen consisting of the cells from the original colonies to a plate consisting of a selective medium with a membrane filter allows the cells to grow on the filter. After the cell growth, the membrane gets lifted and processed for cell lysing.

After the bacterial cell lysis, the DNA gets denatured into single-stranded structures. Then the DNA firmly binds to the filter. After placing the filter on a heat-stable bag, the cDNA probes get incubated with cDNA probes. The labeling of the probes with radioactive or a non-radioactive element involves separate steps. First, the DNA gets denatured by boiling. Next, a quick way of cooling it involves ice for producing the single-stranded molecules. The synthetically obtained primers get annealed to DNA. Next, pairing with the hexanucleotide primers takes place. They get elongated by Klenow fragment of DNA polymerase I using radioactively labeled precursors (dNTPs). An example includes P³² labels. A special DNA precursor molecule gets involved in the case of non-radioactive labeling. The precursor involves the use of the digoxigenin dUTP. It also involves usage of chemiluminescent substrates. The labeled DNA molecules get diffused over the filter. Complementary base pairing and hydrogen bonding form DNA-DNA hybrids. The formation of the hybrids occurs between the probe and the colony DNA. Washing the filter with a suitable solution helps to remove any unbound probes. Colonies on the film present as dark spots get detected using an autoradiography technique. Shuttle vectors and expression vectors also help in screening the genomic libraries.

Complementation of the mutations:

This test relies on the expression of the wild-type gene. Complementation test determines whether the two mutant sites are in the same gene. Crossing the two mutant phenotypes results in wild-type progeny leading to complementation. Let us consider an example of a yeast mutant lacking the arginine biosynthesis due to a mutant arg1 gene. The wild-type ARG1 gene synthesizes arginine. A designing a yeast-E coli shuttle vector involves the synthesis of the ARG1 gene product. The genomic library containing the ARG1 gene gets transformed into yeast containing the mutant phenotype, the ARG1 wild-type gene gets expressed. Hence, it results in complementation. Therefore, the ARG1 gene overcomes the functional defect of the arg1 mutant gene. The plasmid isolated from the cells helps in the characterization of the cloned gene.

Complementation test helps in identifying genes in the library. Shuttle vectors are bifunctional vectors. They grow and transform into prokaryotes as well as eukaryotes.

Heterologous and oligonucleotide probes:

A small and a defined nucleic acid (either DNA or RNA) identifying the specific molecules with complementary sequences are known as probes. Depending on the type of labeling, the probes matching their complementary sequences get detected through autoradiography. A probe searches a complementary sequence in a cDNA library, a genomic library, northern blot or in situ hybridization. Depending on a good degree of homology between the probes and the genes, the heterologous probes work. The probes consisting of equivalent genes obtained from other organisms are known as heterologous probes. Example, a mouse probe serves as a probe for a human genome library. It involves working with highly conserved genes. Hence, a heterologous probe is similar to the nucleic acid sequence but not complementary to the nucleic acid. Heterologous probing identifies related genes in the same organism. For example, wheat gliadin cDNA clones serve as probes. It hybridizes to a complementary sequence and a variety of other genes. Another example includes a yeast cytochrome c gene as a probe in identifying probable Neurospora cytochrome c clone in a Neurospora gene library.

Oligonucleotide probes identify genes or cDNA libraries. It is possible to clone genes that lack previous genetic information.

References:

[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2] Recombinant DNA Principles and Methodologies, James Greene

[3] IGenetics, Peter Russell

[4] Genetic Engineering, Verma P.S. & Agarwal V.K.

[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh
[6] Gene cloning and DNA analysis, T.A. Brown

© 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

   

© Copyright, 2018 All Rights Reserved.