What is a recombinant dna technology?

Recombinant DNA technology is a powerful tool used by molecular biology researchers to create hybrid or chimeric DNA to develop pharmaceutical products or study the function and regulation of gene or DNA sequences of interest. The process involves cleaving the DNA sequence of interest and the suitable self-replicating vector (such as plasmid) using the same restriction endonuclease enzyme and joining them together using a molecular glue known as ligase. The process is known as ligation.

The vector is then introduced into a bacterial cell (such as E. coli) or yeast, where their replication and translation machinery is employed to replicate plasmids and synthesize the desired proteins. The technology has successfully produced therapeutic products, such as insulin, vaccines, and growth hormones to treat various human diseases. E. coli or Saccharomyces cerevisiae transformation can be carried out with recombinant plasmid or self-ligated vector. Both kinds of transformants will grow on the selective medium. Thus, it’s necessary to screen all the transformants using an appropriate screening technique. The process enables us to obtain clones that are accurately transformed containing recombinant plasmid and have the inserted or desired DNA sequence in the right orientation.

Explore this article further to learn about various screening techniques available, why they are crucial for research and development, and advancements in the field.

The basics and importance of screening methods in recombinant DNA technology

The process of cloning uses restriction endonuclease enzymes to cut plasmid and gene inserts to generate ends (sticky or blunt ends) that can be ligated. However, the process of ligation can lead to the formation of three kinds of products:

• Self-ligated vector (vector without gene) - In cloning experiments, self-ligation may occur without the insertion of the desired fragment, leading to the formation of empty vectors. This makes it difficult to isolate the desired recombinant clones and reduces cloning efficiency.
• Vector with the gene or DNA sequence of interest (recombinants) - Recombinants enable the production of multiple copies of the gene or sequence for further research, manipulation or cell engineering.
• Gene-gene ligated product – A result of the joining of two different genes, this can lead to the creation of chimeric genes that may exhibit desired or enhanced biological activities.

When these products are introduced into a bacterial or fungal host and plated on appropriate agar media, a mixture of clones, each containing all three products, is formed. Thus, in this regard, screening approaches are needed to identify the correct clone having a vector with the sequence of gene of interest. They differentiate recombinants from non-recombinants.

The screening of genomic and cDNA libraries, containing thousands of different types of clones with different DNA fragments, helps obtain the right clone in a single shot. This saves a sizeable amount of time, effort, resources, and money spent on performing the same experiment repeatedly.

Overall, a cloning experiment's success is determined by the application of a screening approach.

Commonly used screening methods in recombinant DNA technology

Some commonly used screening methods are listed below:

Antibiotic-based screening approach

In this method, recombinants are distinguished by the absence of gene function the vector encodes. For instance, if the pBR322 plasmid vector contains two antibiotic resistance genes, (ampicillin and tetracycline) that enable it to grow on a media containing any of these antibiotics. However, a fault or mutation in any one of the antibiotic resistance genes due to the insertion of the gene of interest makes them sensitive to one antibiotic. For example, if the gene is inserted between the tetracycline gene, the recombinants will be sensitive to tetracycline but will be able to grow on ampicillin.

During the process, the recombinants are grown on an ampicillin-containing media (known as master plate), whose patterns are transferred to a nitrocellulose membrane by gently pressing the membrane on the plate. This creates a replica of the colony distribution as it was in the master plate. The membrane is then placed on a media containing both ampicillin and tetracycline. This led to the growth of only non-recombinants having resistance to ampicillin. Then, the master plate and replica plate are compared to select the colonies having recombinants. The process is known as replica plating.

Chromogenic screening method

It’s one of the old and most commonly used techniques for recombinant screening that distinguishes between recombinant clones and non-recombinants based on color differences between the colonies. It’s commonly known as blue-white screening. The technique utilizes a β-galactosidase enzyme, encoded by the lacZ gene in E. coli, that hydrolyzes X-Gal (an analog for lactose) into 5′-dibromo-4,4′-dichloro-indigo (an insoluble deep blue pigment), forming deep-blue colonies.

The host cells used are mutants for the lacZ gene. The mutation affects the synthesis of the carboxyl end of the β-galactosidase enzyme. The plasmid vector produces only the first 146 amino acids of the β-galactosidase enzyme. Thus, neither the vector nor the host, on their own, can produce a fully functional enzyme. However, when the host carries these recombinant vectors, they synthesize functional β-galactosidase enzymes due to α-complementation.

During the process, the gene of interest is inserted by cleaving at the multiple cloning site (MCS), using a restriction enzyme, between the lacZ gene. After ligation, the vector is transformed into the E. coli host and cultured on an agar gel medium containing a specific antibiotic (for selecting transformants), X-gal, and Isopropyl β-D-1-thiogalactopyranoside (IPTG) that act as inducers for the LacZ promoter. The recombinant colonies are observed in white color due to interruption of the lacZ gene expression and non-functional β-galactosidase enzyme, whereas non-recombinants produce deep-blue color colonies due to the synthesis of functional β-galactosidase enzyme.

Colony PCR

After the blue-white screening, it’s another commonly used screening technique for recombinants. In this method, all the bacterial or fungal colonies from the master plate are pooled in a multi-well plate. The cells are lysed and subjected to PCR along with a set of target DNA-specific primers. Subsequently, individual colonies from the master plate, part of the positive pool, undergo PCR with the same set of primers. The technique will produce a product of known size only in the presence of the insert. In its absence, a product of a different size will be observed on the agarose gel. The method offers more accurate and rapid detection of recombinant target clones and also the orientation of the insert. However, it is only effective for products that are less than 1 KB in size.

Nucleic acid hybridization technique

The technique utilizes the binding efficiency of the probe with the target sequence. The higher the similarity between the sequence of the probe and the target sequence, the higher the percentage of homology. For example, if the sequence and probe come from the same organisms, there will be 100% homology. However, the percentage decreases in case the sequence is from a related but not the same organism. In the process, the DNA/RNA probes are labeled using radioactive (less commonly used) or non-radioactive molecules, such as enzymes, and detected due to some fluorescence or change in color due to reaction with the substrate.

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Advancements in screening methods

Though blue-white screening (such as pBlueScript) has been in use for more than 25 years, nonetheless, this method demands specific bacteria modified with relevant α-subunit gene deletions and X-gal. During cloning, ambiguous results are common, especially when dealing with small inserts. Similarly, the issue with the colony PCR technique approach is the need for highly accurate and precise primers for the reaction. Such limitations of the available technique demand some advanced approaches that reduce multiple screening steps to a single or couple of stages, are effective, and save time.

For example, researchers developed a pRedScript technique (a modified version of the pBlueScript technique) that allows the screening of recombinants using red (non-recombinant) or white color (recombinants). However, the method does not require any special instrument or engineered bacteria like the pBlueScript technique. Similarly, this study describes an ultra-high efficient colony PCR method that can be used with different types of hosts (unlike traditional colony PCR that’s restricted to E. coli strains) is rapid, and can be performed with just a few pieces of equipment.

Thus, researchers are working on improving the traditional technique to build a robust, time-saving, and inexpensive screening approach for large-scale applications.

Conclusion

Specific recombinant clonescan now be isolated and characterized using cDNA and genomic libraries. We now understand prokaryotic and eukaryotic organisms’ genetic material better in terms of their localization within the cells, their structure, organization, regulation, and gene expression.

The effective isolation, identification or recognition, and the subsequent cloning of targeted genes enables recombinant proteins’ expression. This expression is employed for diverse purposes, spanning research, agriculture, and medicine. However, without screening of recombinants, the specific end product cannot be achieved. The screening method helps save time and resources by identifying the right clones in a single experiment.

While traditional methods like blue-white screening and colony PCR have been employed to identify the correct clone, scientists are actively designing advanced high-throughput screening approaches or modifying traditional techniques. This is to establish a robust system that not only accelerates the clone screening process but also minimizes the need for specialized equipment and materials. The overarching goal is to make the screening process more cost-effective, faster, and accessible to researchers.

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