DNA cloning refers to the process of generating multiple copies of a particular DNA segment. DNA and Gene Cloning involves the isolation of a DNA sequence of any species and its insertion into a vector to enable growth without any alteration in the original DNA sequence. Once isolated, molecular clones can be used to generate multiple copies of DNA for the analysis of gene sequences and / or to express proteins obtained for the study or utilization of protein function. These clones can be manipulated and mutated in vitro to alter the expression and function of a protein.
Further, DNA contains some specific patterns of nucleic acid sequences, for instance, transcription factors as DNA binding sites for a regulatory protein, known as motifs. There are some motifs, such as hairpin loops which hinder the usability of a gene. Elimination of these bad motifs helps in circumventing problems such as poor PCR to an extent.
Genes can be categorized into three different categories based on their length and complexity.
Gene Strands: The general characteristics of a gene strand include the linear and dsDNA fragments with their length ranging between 20-99 bp.
Standard Genes: Standard genes are the genes whose variations are controlled by one single gene. These genes possess the following characteristics:
GC content: Moderate (40-65%)
No critical hairpin loops
Not more than 20 bp repeats
Not more than 20bp hairpin stretches
Complex Genes: The interaction of a gene with external factors, either environmental or genetic, influences the normal trait, making it complex. Complex genes include the following characteristics:
GC content: Very low or very high
Long repeat sequences
More number of secondary structures
Challenges Associated with In-house DNA and Gene Cloning
The process of gene cloning not only involves the creation of multiple copies of a gene or any other cell component, but also replication of any gene post its replacement within the same DNA. This has enabled several researchers to study genetic disorders more precisely and closely. In addition to this, gene cloning has been observed to be a potential solution to organ scarcity, which is currently one of the major concerns surrounding human lives, by cloning organs in small animals. However, there are several challenges and complications associated with the successful DNA / gene cloning including gene toxicity, unstable DNA elements, insert size or large vector, and the presence of DNA secondary structures.
Further, there are ethical issues as well, along with reproductive and mutation challenges as well associated with DNA cloning and gene cloning.
DNA cloning is a largely accepted technique, however, it has been regulated carefully. Within DNA cloning, both therapeutic and reproductive cloning raise an important ethical issue, especially regarding the potential use of these techniques in humans. The freedom to manipulate the genetic material and adulterate human species may lead to adaptive malfunctions. There are many conflicts associated with reproductive cloning. These can be related to long-standing societal values about human dignity, possibly infringing upon principles of individual freedom, autonomy and identity.
Possible Solution: Outsourcing of DNA Cloning Services and Gene Cloning Services
Given the fact that more than 140,000 cDNA clones can be found across mouse, human and rat population, the expression of multiple targets including transmembrane proteins, receptors, toxins, ion channels require optimization. However, this often affects protein purification and expression. The process of DNA cloning / gene cloning needs to be monitored closely to ensure high-quality and stable finished product. Owing to all these challenges involved in the DNA cloning and gene cloning, the outsourcing of various operations involved in the process DNA cloning is being actively preferred by stakeholders engaged in this domain. Presently, several companies offer DNA cloning services and gene cloning services, making recombinant DNA that can be used by academic and biopharmaceutical industries.
Benefits Offered by Service Providers
The various benefits offered by DNA and gene cloning service providers are:
The technical expertise of DNA and gene cloning service providers helps in creating custom DNA constructs for various research and downstream based applications. Further, the labor and reagent cost incurred in cloning procedures is reduced with customized cloning. Along with this, the high quality DNA clones and gene clones are customized as per the need and requirement.
DNA Cloning Methods Used by Service Providers
The various DNA cloning methods have been proven to be useful in redesigning organisms and mending them with new abilities, specifically during the pandemic. Cloning methods used by various DNA and gene cloning service providers have been described below:
It is a traditional method of DNA and gene cloning. The basic steps involved in the molecular cloning are:
Isolation: A gene / DNA sequence is isolated from any species, also known as insert
Insertion: Incorporation of inserts into cloning vector, without altering the original DNA sequence
Propagation: Transformation of recombinant plasmids into bacteria or any other suitable host for propagation
Screening: Selection of hosts containing the intended recombinant plasmid
2. Genetic Synthesis: It includes in-vitro synthesis of any DNA sequence or gene without the involvement of initial template. Since there is no involvement of DNA template, it is possible to make a complete synthetic DNA, without any restriction of nucleotide sequence and size. This involves two steps:
Solid-phase DNA synthesis: This step produces oligonucleotide fragments, generally a stretch of 200 base pairs. It is also known as DNA printing.
Ligation: This step involves ligation of these short stretches of DNA / gene produced by solid-phase DNA synthesis.
3. Custom Cloning: Based on the uncertainties and complexities involved in the process, various end-users including academic and research institutes, pharmaceutical and biotechnology companies seek services related to DNA and gene cloning / recombinant DNA specific to their needs.
Future Perspectives of DNA Cloning
The COVID 19 pandemic has had a significant impact on the DNA cloning market. Several biopharmaceutical players have collaborated and contributed to the research and development of different products, such as DNA cloning kits, vaccines and treatment solutions to combat COVID 19. It is worth noting that cell cloning activities, such as expression of recombinant DNA, proteins and mutagenesis have been attracting significant attention to this market. The advent of innovative cloning technology has opened the doors to more sophisticated applications, such as epigenetic modification, identification and mapping, and high-throughput assembly of combinatorial libraries. Despite several advancements, there are various challenges associated with process related to DNA cloning and gene cloning, for which stakeholders are leveraging the expertise of various contract service providers offering development and manufacturing services.