Genetic Engineering Tools: Precision in DNA Innovation –  

Recombinant DNA technology relies on a suite of powerful tools that enable precise manipulation of genetic material. Tools facilitate the creation of genetically modified organisms and the production of desired proteins, advancing diverse applications in biotechnology.

How do important Genetic Engineering tools, such as vectors and restriction enzymes work together to drive Progress?

• Genetic engineering or recombinant DNA technology can be accomplished only if we have the key tools, i.e., 
  • Restriction Enzymes, 
  • Polymerase Enzymes, 
  • Ligases, 
  • Vectors and 
  • The Host Organism.

Crucial role of restriction enzymes in Genetic Engineering:

• Isolation of Bacteriophage-Restriction Enzymes in 1963: In 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated. 
• Function of Restriction Endonucleases: One of these added methyl groups to DNA, while the other cut DNA. 
  • The latter was called restriction endonuclease. 
    • Nuclease Family: Restriction enzymes belong to a larger class of enzymes called nucleases. 
    • Types: Exonucleases and Endonucleases in Genetic Engineering: These are of two kinds; exonucleases and endonucleases. 
  • Exonucleases: It removes nucleotides from the ends of the DNA.
  • Endonuclease: It makes cuts at specific positions within the DNA. 
    • Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. 
    • Precision Cutting in Genetic Engineering: Role of Restriction Enzymes: Once it finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones.

• Precision Cuts: Mechanism of Restriction Enzymes: Restriction enzymes cut the strand of DNA a little away from the center of the palindrome sites but between the same two bases on the opposite strands. 
  • This leaves single-stranded portions at the ends. 
  • There are overhanging stretches called sticky ends on each strand. 
• Naming:  Hydrogen Bonds and the Role of DNA Ligase: These are named so because they form hydrogen bonds with their complementary cut counterparts. 
  • Facilitate Enzyme: This stickiness of the ends facilitates the action of the enzyme DNA ligase. 
    • DNA Recombination: Role of Restriction Endonucleases in Genetic Engineering Restriction endonucleases are used in genetic engineering to form ‘recombinant’ molecules of DNA, which are composed of DNA from different source
• Specific Requirement: DNA Fragments with Sticky Ends and Ligases: When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘sticky-ends’ and these can be joined together (end-to-end) using DNA ligases.

How do Genetic Engineering Tools use DNA fragmentation to create Recombinant DNA?

• Genetic Engineering Tools: Gel Electrophoresis in DNA Fragmentation Analysis: The cutting of DNA by restriction endonucleases results in fragments of DNA. 
  • These fragments can be separated by a technique known as gel electrophoresis. 
• DNA Separation through Electric Fields in Genetic Engineering: Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. 
• Agarose Gel: Significance in Genetic Engineering Tools: Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from seaweeds. 
• Size-Dependent DNA Separation: The DNA fragments separate according to their size through the sieving effect provided by the agarose gel.
  • Hence, the smaller the fragment size, the farther it moves. 
• Elution: The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. 
  • This step is known as elution. 
  • The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

How do Genetic Engineering tools like Plasmids and Bacteriophages multiply alien DNA?

• Role of Plasmids and Bacteriophages in bacterial cells: They have the ability to replicate within bacterial cells independent of the control of chromosomal DNA. 
• Proliferation of Bacteriophages: Because of their high number per cell, they have very high copy numbers of their genome within the bacterial cells. 
• DNA Multiplication Process: If we are able to link an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal to the copy number of the plasmid or bacteriophage.

Mechanism to facilitate cloning into a vector: Enhancing Replication, Selection, and Expression:

• Role of Origin of replication (ori): This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. 
  • This sequence is also responsible for controlling the copy number of the linked DNA. 
• Selectable marker in Genetic Engineering:: In addition to ‘ori’, the vector requires a selectable marker, which helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. 
• Cloning sites: Simplifying Selection and Enhancing Expression: The recognition sites on plasmids are known as cloning sites.
  • Few Site: In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes. 
    • The presence of more than one recognition site within the vector will generate several fragments, which will complicate gene cloning. 
  • Gene Expression: In the case of two antibiotic resistance genes, one antibiotic resistance gene helps in selecting the transformants, whereas the other antibiotic resistance gene gets ‘inactivated due to insertion’ of alien DNA, and helps in the selection of recombinants. 
  • Cumbersome Process: The selection of recombinants due to the inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics. 
  • Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. 
• In this, a recombinant DNA is inserted within the coding sequence of an enzyme, β-galactosidase. 
  • This results in the inactivation of the gene for the synthesis of this enzyme, which is referred to as insertional inactivation. 
• Vector Strategies in Gene Cloning in plants and animals: 
  • Agrobacterium tumefaciens: It is a pathogen of several dicot plants that is able to deliver a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumour and direct these tumour cells to produce the chemicals required by the pathogen. 
  • Retrovirus: Similarly, retroviruses in animals have the ability to transform normal cells into cancerous cells. 
    • Retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells. 
  • So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a bacterial, plant or animal host (where it multiplies).

How can Genetic Engineering Tools transform host cells with Recombinant DNA?

• DNA is a hydrophilic molecule and cannot pass through cell membranes. 
• In order to force bacteria to take up the plasmid, the bacterial cells must first be made ‘competent’ to take up DNA. 

Diverse Strategies for Introducing Recombinant DNA:

• From Calcium treatment to Gene Guns: Treating them with a specific concentration of a divalent cation, such as calcium, increases the efficiency with which DNA enters the bacterium through pores in its cell wall. 
• Incubation for Recombinant DNA Uptake in Bacteria: Recombinant DNA can be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 420C (heat shock) and then putting them back on ice. 
  • This enables the bacteria to take up the recombinant DNA. 
• Direct Method: Precision Gene Transfer:  In micro-injection, recombinant DNA is directly injected into the nucleus of an animal cell. 
• Gene Gun and disarmed pathogens for DNA transfer: In another method, suitable for plants, cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA in a method known as biolistics or gene gun. 
  • The last method uses ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.

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