Recombinant DNA Technology: Gene Cloning

Recombinant DNA Technology: Gene Cloning

1. Introduction of Gene Cloning

Choosing and creating specific strains of cells is rooted in the understanding that a cell’s inherent abilities are determined by its genes. Genes are composed of DNA, which guides their effects on how the cell behaves. This process involves two steps: transcription and translation. DNA is replicated with high precision through a process called semiconservative replication. This accurate replication ensures that genes are passed down from parents to their offspring without alterations. This reliability is what maintains consistent genetic traits over many generations.

However, there is a natural occurrence of infrequent changes in genes, known as spontaneous mutations, which happen at a rate of around 1 in every 10,000 to 1 in every 1 million genes per generation. These mutations are responsible for the various inheritable differences seen among living organisms. Essentially, mutations introduce diversity, which is then explored through a process called selection during the development of new strains. This selection helps us harness and make use of the diverse traits that arise due to these mutations.

The ways genes naturally move around can be different in how wide they can go and how specific they are. Here are some details about this:

a-The natural methods of gene transfer can be a bit messy. This means that getting the exact combination of genes we want is not easy. We have to use effective methods to pick out the right genes.

b-The movement of genes between different species is not very flexible. This depends on whether the species can reproduce together (through sex) or if a virus can infect them (through a process called transduction). These rules make it hard for genes to move between different types of organisms.

But, during the course of evolution, genes seem to have broken these rules sometimes. For example, genes from bacteria have managed to become part of the human genetic code, even though bacteria and humans are very different from each other, similarly many plant viruses have their genome as a part of the parent plant genome. 

People want to recreate faraway gene transfers in a controlled way and at a rate that’s practically useful. This is done using something called recombinant DNA technology. Here’s how it works with more details:

• First, we pick out the gene we want and make many copies of it.
  
  
• Then, we put this gene into another living thing, which we call a “host.”

We do this for two main reasons:

• The first reason is to make a lot of the protein that the gene codes for. We can then take this protein out and use it.
  
  
• The second reason is to change the way the host organism behaves by using the gene. This change makes the host more useful in some way.

So, in the end, we can either get a useful protein from the gene or change the behavior of the host organism to make it more valuable.

There are three ways to get many copies of a gene you want. Let’s go into more detail:

Figure: Methods to produce multiple copies of a gene

• Gene Cloning: This is the basic method used to make the first set of gene copies. It’s like making a copy of the gene to study it.
  
  
• Polymerase Chain Reaction (PCR): Once we know the sequence of a gene (at least parts of it on both ends), we can use PCR. PCR makes it much easier, faster, and cheaper to make copies of genes. It’s like making lots of copies of a specific section of the gene.
  
  
• Chemical Synthesis and PCR Combo: When we know the entire sequence of a gene or even the sequence of the protein it makes, we can either make the gene using chemicals or by combining PCR with chemical methods. Making a gene with chemicals lets us change the sequence a lot. This is useful when we want to change the gene a lot.
  

In this article, we’re focusing on gene cloning. In the next articles, we’ll talk about other things like restriction enzymes, making genes with chemicals, and using PCR.

2. What is a Recombinant DNA Molecule?

A recombinant DNA molecule is made by putting together pieces of DNA from different sources, usually different living things. Here’s how it works with more details:

• First, we take a small piece of DNA called a vector (like a tool) that can hold other DNA.
  
  
• Then, we add the DNA piece we want to study or use into this vector. This makes a recombinant DNA molecule.
  
  
• We use special tools, like scissors called restriction enzymes, to cut the DNA into smaller parts. And then, we use another tool, like glue called ligation, to put these parts together in the right way.
  

This way, we can create a new DNA molecule that has a gene from one living thing mixed with control parts from another living thing. This new gene is like a mix, and we call it a “chimeric gene.” Having the ability to make recombinant DNA has given us the power to make new combinations of genes to fit our specific needs. It’s like tinkering with building blocks to create something new and useful.

Figure: Construction of a recombinant plasmid DNA molecule (Image Source: Minestrone Soup at English Wikipedia, CC BY-SA 3.0 <http://creativecommons.org/licenses/by-sa/3.0/>, via Wikimedia Commons)

Recombinant DNA molecules are created for three main reasons, like having three goals:

1. Making Many Copies of Specific DNA: Sometimes we need lots of copies of a certain piece of DNA for study.
   
   
2. Getting Lots of Protein: Other times, we want to make a lot of the protein that a gene produces.
   
   
3. Putting Genes into Organisms: And in some cases, we want to add a gene into an organism’s genetic material so it starts working there.
   

For the last two goals, it’s important to first make a bunch of copies of the gene we’re interested in. To do this, we use something like a tool that can copy DNA – it’s called a vector. The most common vectors are like tiny self-replicating machines, and they’re either bacterial plasmids or DNA viruses.

So, here’s the process in a nutshell: We gather pieces of DNA from different places, put them together using these vectors, and this whole process is what we call recombinant DNA technology (RDT). It’s like assembling a puzzle with DNA pieces from different places, and the end result can help us do all kinds of assortments in biology.

The little vehicles (vectors) that carry the DNA pieces we want to study, called “DNA inserts,” are put into a helpful living thing, usually a bacterium. This living thing is called a “host,” and the process is called “transformation.” We then pick and multiply the changed host cells. The special DNA, the recombinant DNA (which is the vehicle plus the DNA piece), in these clones copies itself, either at the same time as the host cell or on its own. The gene in this special DNA might or might not make something important, like a protein.

The step where we change a host with this special DNA and then multiply it is called “DNA cloning” or “gene cloning.” Sometimes, people use these terms to talk about both making this special DNA and growing it in the host.

A “clone” is a bunch of offspring from one single living thing or cell. Making a clone is called “cloning.” In a clone, everyone has the same genetic code, just like the original. This means the DNA in all the members of a clone is exactly the same, even for this special DNA. This is how gene or DNA cloning helps us make a lot of copies of a gene or DNA piece.

People often use the term “recombinant DNA technology” to talk about gene or DNA cloning in the bigger sense. And another common term for these activities is “genetic engineering.” It’s like making special changes in the genes to do specific things.

3. Steps in Gene Cloning

• Getting the DNA Pieces: First, we gather and separate the pieces of DNA we want to work with. This is like collecting puzzle pieces.
  
  
• Combining DNA: Then, we put these DNA pieces into a special tool, called a vector, to create a new kind of DNA called recombinant DNA. Think of it like creating a new recipe by mixing ingredients.
  
  
• Adding the New DNA to a Living Thing: We take this new DNA and add it to a living thing, usually a bacterium like E. coli. This living thing is called the host, and we call this step “transformation.”
  
  
• Picking the Changed Cells: We choose the transformed cells from the host, the ones that have taken up the new DNA. Then, we find the clone with the specific DNA piece we want. A clone is like a group of identical twins.
  
  
• Making More DNA: The new DNA piece starts to copy itself and do its job inside the host cell. This is like having a copied recipe and following it to cook the same dish.
  
  
• Using the New DNA in Other Living Things: If needed, we can move this DNA into another living thing and make it work there too. It’s like sharing the recipe with other cooks.

So, gene cloning is like assembling a puzzle by collecting DNA pieces, making a new DNA recipe, putting it into a living thing, choosing the right cells, making copies of the new DNA, and sometimes sharing it with other living things.

Figure: Steps for Gene Cloning

4. Tools For Gene Cloning

4.1. Restriction Endonucleases

Also known as molecular scissors, are specialized enzymes that can accurately cut DNA at specific recognition sites within the DNA sequence. These sites are typically palindromic in nature. By making targeted cuts, restriction endonucleases play a pivotal role in genetic engineering, allowing scientists to create DNA fragments with known sequences. These fragments can be inserted into other DNA molecules, such as vectors, during gene cloning, enabling the development of recombinant DNA molecules containing desired genes. Derived from bacteria, these enzymes serve as a defense mechanism against viruses by cleaving viral DNA. Their diverse recognition sequences offer researchers a variety of tools for precise DNA manipulation, revolutionizing genetic engineering and facilitating advancements in biotechnology.

Figure: The HindIII restriction nuclease cutting at specific restriction site (5′ A^AGCTT 3′). Green arrows indicate sticky ends. (Image Source: Helixitta, CC BY-SA 4.0 <https://creativecommons.org/licenses/by-sa/4.0>, via Wikimedia Commons)

4.2. Insert Molecule

It refers to the DNA fragment of interest that is incorporated into a vector during gene cloning. This insert can contain specific genes, regulatory elements, or other genetic information. By joining the insert with a vector, researchers create a recombinant DNA molecule, which can be introduced into host cells. The insert’s characteristics determine the resulting properties of the genetically modified organism. ‘Insert’ molecules are pivotal in RDT, allowing scientists to manipulate and transfer desired genetic information, enabling the production of organisms with novel traits for various practical applications.

4.3. Vector Molecule

It serves as a carrier that helps transfer desired DNA fragments into host organisms. Vectors are usually small, self-replicating DNA molecules like bacterial plasmids or viruses. These vectors are modified to contain essential components: an origin of replication for reproduction, selectable markers for identifying successful transformations, and sites where DNA can be inserted. By inserting the DNA fragments into vectors and then introducing the vectors into host cells, researchers can clone and manipulate genes, enabling the production of recombinant DNA molecules with specific traits for various biotechnological applications.

4.4. Ligase Enzyme

It plays a critical role by functioning as molecular glue, sealing the gaps and nicks in DNA strands. Derived from sources like E. coli infected with phage T4, DNA ligase forms phosphodiester bonds between adjacent nucleotides in DNA, joining DNA fragments together. This process is essential when creating recombinant DNA molecules, where DNA fragments from different sources are combined into vectors. Ligase ensures that these fragments are correctly linked, allowing scientists to engineer genes and manipulate DNA sequences, ultimately facilitating the production of genetically modified organisms and other biotechnological advancements.

4.5. Host Organism

It refers to the living entity, often a bacterium like E. coli, used to carry and multiply recombinant DNA molecules. These host organisms are chosen for their ability to self-replicate the recombinant DNA and generate many identical copies. By introducing the recombinant DNA into the host, scientists can harness its replication machinery to clone and express genes of interest. The host provides a controlled environment for the DNA to reproduce, enabling the production of large quantities of specific gene products or proteins. This vital step allows RDT to create genetically modified organisms with desired traits.

4.6. Reverse Transcriptase

It is a pivotal enzyme that converts RNA into DNA. This enzyme plays a crucial role in creating complementary DNA (cDNA) from messenger RNA (mRNA) templates. This process is called reverse transcription and is fundamental when working with genes that are transcribed from RNA viruses or when studying eukaryotic gene expression. Reverse transcriptase allows researchers to produce DNA copies of specific RNA molecules, which can then be integrated into recombinant DNA vectors for further manipulation and study.

4.7. Alkaline Phosphatase

It is an enzyme with significant importance, often used to manipulate DNA. By removing phosphate groups from DNA molecules, this enzyme prevents self-ligation of vector molecules without inserts. This step ensures that only vectors containing desired DNA fragments are used for subsequent DNA manipulations, enhancing the efficiency of gene cloning. Alkaline phosphatase treatment is a crucial technique in RDT as it enables researchers to prepare pure and specific DNA samples, facilitating the successful creation of recombinant DNA molecules for various biotechnological applications.

4.8. T4 Polynucleotide Kinase

Is a vital enzyme used to modify DNA and RNA molecules. By adding phosphate groups to the 5′ ends of nucleotide chains, this enzyme enhances the ability of DNA and RNA to be linked together. This step is crucial for various applications, such as labeling DNA or RNA probes used in genetic studies or ensuring effective ligation of DNA fragments during gene cloning procedures.

4.9. S1 Nuclease

It is a significant enzyme employed to study and manipulate DNA and RNA molecules. This enzyme is particularly useful in detecting single-stranded regions in DNA or RNA molecules. By cleaving the exposed single-stranded segments, S1 nuclease aids in analyzing nucleic acid structures and interactions, essential for gene mapping, sequencing, and hybridization studies. Its ability to selectively degrade single-stranded regions while sparing double-stranded regions makes S1 nuclease an invaluable tool in RDT.

4.10. Klenow fragment

It is a crucial enzyme derived from DNA polymerase I. It possesses the ability to synthesize DNA strands by adding nucleotides to a DNA template. Often utilized in DNA sequencing, site-directed mutagenesis, and labeling DNA probes, the Klenow fragment has a unique property—its inability to initiate DNA synthesis without a primer-template junction. This characteristic allows researchers to generate complementary DNA copies from DNA or RNA templates.

4.11. Lambda Exonuclease

It is a significant enzyme used for DNA modification and manipulation. Derived from the bacteriophage Lambda, this enzyme possesses the ability to degrade one strand of double-stranded DNA, moving in a 5′ to 3′ direction. This process helps generate single-stranded DNA fragments with controlled lengths, which are essential for various applications such as creating DNA probes, generating single-stranded templates for sequencing, and studying DNA-protein interactions. Lambda exonuclease plays a vital role in RDT by enabling precise DNA fragment preparation and enhancing our ability to analyze and modify genetic material for a range of biotechnological and research purposes.

4.12. Exonucleases from Escherichia Coli

These are enzymes used to modify and manipulate DNA molecules. These enzymes work by removing nucleotides from the ends of DNA strands in a stepwise manner. For instance, Exonuclease III is known for removing nucleotides from 3′ ends, while Exonuclease I function at the 5′ end. These exonucleases are essential tools in RDT, used for various applications like removing unwanted sequences, generating cohesive ends for DNA fragments, and preparing templates for DNA sequencing.

4.13. Terminal deoxynucleotidyl Transferase (TdT)

It is a crucial enzyme used to add nucleotides to the 3′ ends of DNA molecules. Unlike other DNA polymerases, TdT doesn’t require a template, allowing it to add nucleotides in a template-independent manner. This unique property makes TdT valuable for adding specific sequences to DNA ends, a process often used in creating labeled DNA probes, tailing DNA fragments for cloning, and generating diverse DNA libraries. TdT’s ability to add nucleotides to single-stranded overhangs contributes to the customization of DNA fragments for various research and biotechnological applications in RDT.

4.14. Linker and Adaptor Oligonucleotide Sequences

These are short, synthetic DNA molecules designed to facilitate the manipulation and cloning of DNA fragments. Linkers are typically double-stranded molecules with cohesive ends that can be ligated to the ends of DNA fragments, enabling their insertion into vectors. Adaptors, on the other hand, are single-stranded molecules that possess specific complementary sequences to hybridize with DNA fragments. These sequences are then converted into double-stranded DNA, allowing subsequent cloning steps. Linker and adaptor sequences serve as versatile tools, aiding in the construction of recombinant DNA molecules, the preparation of DNA libraries, and the amplification of specific DNA regions via techniques like PCR.

Figure: Cloning of foreign DNA into bacterial plasmid having lactose metabolizing and ampicillin resistance enzymes (Image Source: CNX OpenStax, CC BY 4.0 <https://creativecommons.org /licenses/by/4.0>, via Wikimedia Commons)

5. Significance of Gene Cloning

The significance of gene cloning in modern biology and biotechnology is profound. Gene cloning, enabled by Recombinant DNA Technology (RDT), has revolutionized our ability to manipulate and understand genetic material. Its importance can be outlined as follows:

Understanding Gene Function: Gene cloning allows scientists to study individual genes in isolation, helping to decipher their functions and roles in various biological processes. By cloning and modifying genes, researchers can uncover their effects on development, health, and diseases, paving the way for targeted therapies and treatments.

Biomedical Research: Cloning genes associated with diseases helps scientists analyze the genetic basis of disorders. This knowledge is invaluable for diagnosing conditions, developing predictive tests, and designing potential interventions. Cloning also enables the creation of disease models, aiding drug testing and development.

Biotechnology Applications: Gene cloning is the foundation of biotechnology. It enables the production of essential proteins, hormones, enzymes, and antibodies on a large scale. These products have applications in medicine, agriculture, and industry, benefiting healthcare, food production, and manufacturing.

Creating Transgenic Organisms: Gene cloning facilitates the introduction of foreign genes into organisms, creating transgenic organisms with novel traits. These genetically modified organisms (GMOs) can exhibit increased disease resistance, improved nutritional content, and enhanced growth, contributing to sustainable agriculture and resource management.

Evolution and Taxonomy Studies: Cloning genes across different species helps researchers understand evolutionary relationships and genetic diversity. This knowledge contributes to taxonomy studies and sheds light on the interconnectedness of all life forms.

Gene Therapy: Gene cloning has paved the way for gene therapy, a promising avenue for treating genetic disorders by replacing or correcting defective genes. This approach has the potential to revolutionize the treatment of inherited diseases.

Forensic Science: Cloning specific DNA sequences assists in forensic investigations by enabling the identification of individuals through DNA profiling. This is crucial in criminal investigations and disaster victim identification.

Environmental Applications: Cloning genes involved in environmental processes allows scientists to monitor pollution levels and assess the impact of environmental changes on ecosystems.

Basic Research: Gene cloning plays a vital role in unraveling fundamental biological questions, leading to discoveries that shape our understanding of life itself.

In essence, gene cloning is a versatile tool that permeates multiple facets of science and technology. Its applications range from basic research to applied biotechnology, revolutionizing fields as diverse as medicine, agriculture, ecology, and industry. As technology advances, the significance of gene cloning continues to expand, offering unparalleled insights and opportunities for innovation.

6. Conclusion

The above discussions have provided insight into a range of essential tools crucial for gene cloning in Recombinant DNA Technology (RDT). However, this is just the beginning. In forthcoming articles, we will delve deeper into other significant tools, including restriction enzymes, polymerase chain reaction (PCR), gel electrophoresis, and DNA sequencing techniques. Each of these tools plays a distinct role in the intricate process of gene manipulation and cloning, further enhancing our understanding of genetic engineering and its applications across biotechnology and scientific research. Stay tuned for an exploration of these powerful tools that collectively shape the landscape of modern molecular biology.

7. References

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Howe, C., 2007. Gene cloning and manipulation. Cambridge University Press.

Schamhart, D.H. and Westerhof, A.C., 1999. Strategies for gene cloning. Urological research, 27, pp.83-96.

Wong, D.W., 2006. The ABCs of gene cloning. Springer.

Brown, T.A., 2020. Gene cloning and DNA analysis: an introduction. John Wiley & Sons.

Zuo, P. and Rabie, A.B.M., 2010. One-step DNA fragment assembly and circularization for gene cloning. Current Issues in Molecular Biology, 12(1), pp.11-16.

Mao, Z., Shay, B., Hekmati, M., Fermon, E., Taylor, A., Dafni, L., Heikinheimo, K., Lustmann, J., Fisher, L.W., Young, M.F. and Deutsch, D., 2001. The human tuftelin gene: cloning and characterization. Gene, 279(2), pp.181-196.

8. FAQ’s on Gene Cloning