Genetic engineering, also known as recombinant DNA technology, is a cutting-edge scientific discipline that involves the precise manipulation of genes using laboratory techniques. It enables scientists to transfer genes with known functions from their original locations into cells using suitable vectors. Once transferred, these genes can replicate normally and be passed on to subsequent generations of cells, allowing for the production of multiple copies of the same gene. Genetic engineering has revolutionized the fields of biotechnology, medicine, agriculture, and more, as it offers a wide range of practical applications, such as producing cloned DNA fragments, generating large quantities of proteins, and integrating genes into host organisms. This technology holds the promise of advancing scientific research and providing innovative solutions to various real-world problems.
What Is Genetic Engineering?
Genetic engineering, also known as recombinant DNA technology, is a scientific discipline that involves the manipulation of genes using in vitro processes. In this process, a gene with a known function can be transferred from its original location into a cell using a suitable vector. The transferred gene can then replicate normally and be passed on to the next generation of cells. This allows for the production of multiple copies of the same gene.
The development of procedures for reintroducing foreign DNA fragments into a bacterium has led to the evolution of new technologies, including recombinant DNA technology, gene cloning, and genetic engineering.
Genetic engineering or gene manipulation is a relatively new field of science that focuses on altering the hereditary apparatus of living cells. It involves highly sophisticated techniques for artificially synthesizing or isolating specific genes or DNA fragments, integrating them into suitable vectors, and cloning these vectors in other organisms, referred to as hosts. This technology has several practical applications, including:
- Producing multiple copies of cloned DNA fragments (cDNA): This allows for the amplification of specific genetic material for further study or manipulation.
- Generating large quantities of proteins produced by cloned genes: By cloning and expressing genes in host organisms, scientists can produce proteins of interest for various applications, such as medical research, pharmaceuticals, or industrial processes.
- Integrating genes into the chromosomes of the host organism (gene transfer): This process can be used to repair or replace defective genes within an organism or introduce new genetic traits.
Genetic engineering has opened up numerous possibilities for scientific research and has practical applications in fields such as biotechnology, medicine, agriculture, and more. It allows scientists to modify and manipulate the genetic material of organisms, which can lead to the development of new technologies and solutions for various problems.
Major Contribution to The Development of Recombinant DNA Technology
| Person | Contribution |
|---|---|
| Miescher | Isolated DNA for the first time |
| Avery | Provided evidence that DNA carries genetic info |
| Griffith | Bacterial transformation |
| Watson and Crick | Proposed the double-helical model of DNA |
| Kornberg | Discovered DNA polymerase |
| Arber | First evidence for the existence of DNA-restriction nucleases |
| Boyer, Cohen, Berg | Developed DNA cloning techniques |
| Restriction enzymes | Used to break DNA molecules |
Various Tools for Recombinant DNA Technology
Genetic engineering involves the use of various biological tools for the manipulation of genetic material and cells. There are three primary types of biological tools used in genetic engineering: enzymes, cloning vectors, and competent hosts.
Enzymes
Various specific enzymes are employed in genetic engineering, including recombinant DNA technology. Lysing enzymes play a crucial role in this process by facilitating the breakdown of cell walls to extract DNA for recombinant DNA experiments. Typically, lysozyme is utilized to dissolve bacterial cell walls, enabling the extraction of genetic material for further manipulation and recombination.
Restriction enzymes belong to a larger class of enzymes known as nucleases. They are categorized into three types: exonucleases, endonucleases, and restriction endonucleases.
Exonucleases remove nucleotides from the terminal ends of DNA strands.
Endonucleases make cuts at specific positions within DNA, without cleaving the ends.
Restriction endonucleases recognize specific base sequences in palindromic regions of DNA and cut at those sites. However, type I and type III restriction endonucleases are not commonly used in recombinant DNA technology due to their complex requirements, including ATP and S-adenosylmethionine, for cleavage and recognition of specific sites within DNA.
Type II restriction endonucleases are enzymes that rely on Mg²+ restriction activity. They find application in genetic engineering to facilitate DNA restriction. These enzymes are valuable in genetic engineering due to their ability to recognize and cleave specific DNA sequences, typically spanning 4 to 8 nucleotides, in vitro. They are instrumental in gene manipulation.
Type III restriction endonucleases are comprised of two distinct subunits and rely on ATP, Mg², and S-adenosyl methionine for their restriction activity. These enzymes occupy an intermediate position between Type I and Type II enzymes, as they exhibit both restriction and methylation capabilities. Due to their dual functions, they are not commonly employed in recombinant DNA technology.
The restriction enzymes cut DNA molecules by cleavage in any one of the following two styles :
(i) End Cleavage Style: In this cleavage style, the target DNA sequence is asymmetrical. The cuts made in the two strands of DNA are staggered and separated by several nucleotides, resulting in the creation of complementary single-stranded protruding ends. These unmatched, protruding ends are commonly referred to as sticky or cohesive ends.
(ii) Blunt End Cleavage Style: Enzymes such as Alu I, Hae III, Hind II, and Sma I belonging to the class of restriction enzymes cleave both strands of DNA at the same location. Consequently, the DNA fragments generated have blunt ends. This characteristic allows for the precise fragmentation of DNA of various types, depending on the specific restriction enzyme used. It also facilitates the joining of two well-defined fragments without the introduction of any extra material between them.
DNA ligases, often referred to as genetic gum, are enzymes responsible for creating phosphodiester bonds between adjacent nucleotides, thus covalently connecting two separate segments of double-stranded DNA. The ligase enzyme’s function is contingent upon the presence of a phosphate group at the 5′ carbon of one nucleotide and a hydroxyl group at the 3′ carbon of the neighboring nucleotide, which results in the formation of a 5′ bond between the two nucleotides.
Alkaline phosphatase is employed to eliminate the phosphate group located at the 5′ end of a DNA molecule, resulting in a free 5′ hydroxyl group. This application serves to prevent unintended self-ligation of vector DNA molecules during genetic engineering procedures.
Synthesizing enzymes play a crucial role in the generation of DNA strands from appropriate templates. These enzymes come in two main categories: reverse transcriptase, which synthesizes DNA using mRNA as a template, and the other type polymerizes DNA synthesis on a DNA template or complementary DNA (cDNA).
Additionally, these enzymes are capable of catalyzing either 5’➞ 3′, or 3’➞ 5′ degradation of DNA.
Cloning Vectors (Vehicle DNA)
Cloning vectors are DNA molecules capable of carrying foreign DNA segments and replicating within the host cell. These vectors can take the form of plasmids, bacteriophages, cosmids, phagemids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), transposons, and viruses.
A vector can be defined as a DNA molecule that possesses the following characteristics:
- Autonomously replicates, allowing for the generation of multiple copies within a single host cell.
- Compact and of low molecular weight for increased stability.
- Contains recognition sites for a wide range of restriction enzymes.
- Can be easily isolated and purified.
- Facilitates the straightforward transformation of host cells.
- Has the capacity to integrate either itself or the DNA segment it carries into the host cell’s genome.
- Contains marker genes that enable the selection of transformed host cells.
- Exists in multiple copies within the cell, ensuring more duplicates of inserted DNA sequences.
Plasmid Vectors – Plasmids, initially discovered by William Hays and Joshua Lederberg in 1952, are extrachromosomal, self-replicating, typically circular, double-stranded DNA molecules naturally occurring in many bacteria and certain yeast strains. These plasmid molecules can exist within the host organism as single or double copies or in multiple copies (ranging from 1 to 200 or more).
These naturally occurring plasmids have been adapted for use as vectors in laboratory settings. One of the most widely employed and easily manipulated vectors is the pBR322 vector, which stands as an exemplary plasmid vector. The pBR322 vector was the pioneering artificial cloning vector crafted in 1977 by Boliver and Rodriguez. In the name “pBR322,” ‘p’ signifies that it is a plasmid, ‘BR’ pays homage to Bolivar and Rodriguez, the creators of this plasmid, and ‘322’ is a distinct identifier that sets this plasmid apart from others developed within the same laboratory.
Bacteriophage Vectors: Bacteriophages, which are viruses that infect bacterial cells by delivering their DNA into these cells, possess the unique capability to transfer genetic material from the phage genome to specific bacterial hosts during the bacterial infection process. This remarkable ability has been harnessed to create phage vectors, serving as a valuable tool in genetic engineering. Two notable examples of phages modified for the development of cloning vectors are lambda (λ) and M13 phage.
Cosmid Vectors: Cosmids( = cos + plasmid ) are engineered by combining key features of plasmids with the “cos” sites found in the lambda phage. A basic cosmid vector typically includes a plasmid origin of replication, a selectable marker, well-placed restriction enzyme sites, and the lambda “cos” site. Cosmids are particularly useful for cloning DNA fragments with lengths of up to 45 kilobases (kb).
Bacterial Artificial Chromosome Vectors: Bacterial artificial chromosome (BAC) vectors are constructed using the naturally occurring extra-chromosomal plasmid of E. coli known as the fertility or F-plasmid. A BAC vector is equipped with essential genes responsible for the replication and upkeep of the F-factor, a selectable marker, and a cloning site. These versatile vectors are capable of accommodating DNA fragments ranging from 300 to 500 kilobases (kb) and are frequently utilized in genome sequencing projects.
Yeast Artificial Chromosome Vectors: Recombinant DNA technology employs yeast artificial chromosome (YAC) vectors for the cloning of DNA fragments surpassing 1 megabase (Mb) in size. Consequently, these vectors have been widely utilized in the comprehensive mapping of expansive genomes, such as in the Human Genome Project. YAC vectors include crucial components like telomeric sequences, centromeres, and autonomously replicating sequences from yeast chromosomes. They also feature strategically placed restriction enzyme sites and genes that serve as selectable markers when used in yeast.
Phagemid vectors are a unique type of molecular tool that combines the advantages of both plasmids and phages. They contain a fragment of phage DNA, including its attachment (att) site. Phagemids offer the benefits of plasmids, such as ease of replication and maintenance, while also incorporating the ability to integrate into bacterial chromosomes, akin to the way λ phage DNA inserts itself during the lysogenic phase of the phage life cycle.
Animal and plant viral vectors: Animal and plant viral vectors are viruses that have been modified to serve as tools for delivering foreign DNA into cultured animal and plant cells. These viruses are engineered to harness their natural capacity to attach to cells and initiate infection, making them useful as vectors for introducing foreign genes into eukaryotic cells grown in laboratory cultures.
Transposons as vectors: Transposons, also known as “jumping genes,” are DNA sequences that can change their position within the genome. They have the ability to excise from one genomic location and then insert themselves into another. Transposons are commonly employed as vectors in genetic studies. Notably, in maize, there are well-known transposons called “Ac-Ds elements,” where “Ac” stands for activator and “Ds” stands for dissociator, and these elements are often used for genetic manipulation.
Shuttle vectors are a type of cloning vector designed to function in both eukaryotic cells and Escherichia coli (E. coli) bacterial cells. These vectors are constructed with elements that allow them to replicate and be selected for in both cell types, making them versatile tools for molecular biology and genetic engineering experiments that involve both eukaryotic and prokaryotic systems.
Shuttle vectors typically contain the following key components:
- Multiple Origins of Replication: Shuttle vectors have at least two origins of replication, one that is recognized by eukaryotic cells and another that is recognized by E. coli. This allows the vector to replicate in both types of cells. In eukaryotic cells, these origins are typically derived from plasmids or other eukaryotic DNA elements, while in E. coli, they are derived from bacterial origins of replication.
- Selectable Marker Genes: These vectors also contain selectable marker genes for both eukaryotic and bacterial cells. In eukaryotic cells, the marker gene may confer resistance to a specific drug or selection for some other characteristic (e.g., fluorescence). In E. coli, commonly used markers include antibiotic resistance genes, such as ampicillin or kanamycin resistance.
- Polylinker or Multiple Cloning Site (MCS): A polylinker or MCS is a region within the shuttle vector where researchers can easily insert their DNA of interest. It contains multiple unique restriction enzyme sites, which simplifies the process of cloning DNA fragments into the vector.
The advantage of shuttle vectors is that they allow for the convenient transfer of genetic material between eukaryotic and bacterial systems. For example, you can clone a gene of interest into a shuttle vector in E. coli, propagate it there, and then transfer it to a eukaryotic cell for further experimentation. This is particularly useful when working on gene expression studies or creating recombinant organisms.
Shuttle vectors are commonly used in biotechnology and molecular biology research, especially when working with organisms like yeast, mammalian cells, or other eukaryotic systems. They simplify the process of manipulating and transferring genetic material between different types of cells.
Competent Host
Recombinant DNA transformation is a crucial process in genetic engineering, involving the incorporation of naked DNA fragments from the environment into a host cell’s chromosomal DNA. This integration allows the host cell to express traits controlled by the incoming recombinant DNA. In the context of animal cells, the term “transformation” is often replaced with “transfection.”
A diverse range of host cells is available for genetic engineering purposes, encompassing E. coli, yeast, animal cells, and plant cells. The choice of host cell primarily hinges on the duration and complexity of the cloning experiment. In some instances, particularly for the expression of certain eukaryotic proteins, selecting the appropriate host cell is of paramount importance for the successful creation of recombinant DNA.
Eukaryotic cells often stand as the preferred hosts for working with recombinant DNA. Yeasts, in particular, have seen extensive use in the functional expression of eukaryotic genes due to several inherent advantages. Yeasts represent the simplest eukaryotic organisms, akin to bacteria in their single-celled nature. Furthermore, they are genetically well-characterized, straightforward to cultivate, and amenable to manipulation. They can be cultivated with ease in both small-scale culture vessels and large-scale bioreactors.
In addition to yeasts, plant, and animal cells also serve as viable hosts in gene manipulation experiments and for the expression of recombinant DNA, either within tissue culture systems or as cells within the whole organism. This flexibility allows for the creation of genetically modified plants and animals, expanding the possibilities for recombinant DNA applications in the realm of genetic engineering.
As DNA is inherently hydrophilic, it cannot naturally traverse cellular membranes. Therefore, a necessary step in the process is to render bacterial cells capable of receiving DNA. This is achieved through treatment with a specific concentration of a divalent cation, such as calcium, which enhances the efficiency of DNA entry into the bacterium via pores within its cell wall.
The introduction of recombinant DNA into these cells involves a controlled procedure. First, the recombinant DNA is introduced to the cells while maintaining a low temperature, typically by incubating them on ice. Subsequently, a brief exposure to elevated temperature at around 42°C, commonly referred to as “heat shock,” is employed. This sudden shift in temperature is followed by returning the cells to a cold environment. This precisely choreographed process allows the bacteria to successfully uptake the recombinant DNA, facilitating the integration of the foreign genetic material into the host cells.
Various alternative methods have been devised to introduce recombinant DNA into competent host cells, bypassing the need for carrier molecules. These methods include:
(i)Microinjection: In this approach, foreign recombinant DNA is directly injected into the nucleus of animal or plant cells using fine micro-needles. This precise method enables the targeted introduction of recombinant DNA into host cells.
(ii) Electroporation: In this technique, brief electrical impulses are applied to create temporary pores in the cell membranes of plants, allowing for the incorporation of recombinant DNA molecules. This electrical i facilitates the entry of recombinant DNA into plant cells.
(iii)Gene gun or biolistic: In this method, recombinant DNA is coated onto minuscule pellets made of materials like gold, typically measuring 1-2 micrometers in size. These coated pellets are then propelled into target cells at high velocities. This powerful technique is often used to insert genes into animals, promoting tissue repair in cells near wounds and leading to a reduction in healing time. It’s an innovative means of delivering recombinant DNA to the intended cells without the use of carrier molecules.
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