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.
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[3] IGenetics, Peter Russell
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[6] Gene cloning and DNA analysis, T.A. Brown
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