Modern Biotechnology: Core Techniques & Recombinant DNA Technology

Posted on Sep 15, 2024 in Biology

Two Core Techniques that Enabled the Birth of Modern Biotechnology

Genetic engineering: Techniques to alter the chemistry of genetic material (DNA and RNA) to introduce into host organisms and thus change the phenotype of the host organism.

Maintenance of sterile (microbial contamination-free) ambient chemical engineering processes: To enable the growth of only the desired microbe/eukaryotic cell in large quantities.

Conceptual Development of the Principle of Genetic Engineering

Asexual reproduction preserves the genetic identity of species. Sexual reproduction creates variation and unique combinations of genetic makeup. Traditional hybridization procedures used in plant and animal breeding lead to the inclusion of undesirable genes along with desired genes. The techniques of genetic engineering, which include the creation of recombinant DNA, use of gene cloning, and gene transfer, overcome this limitation and allow us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism.

Three Basic Steps in Genetically Modifying an Organism

  1. Identification of DNA with the desirable gene.
  2. Introduction of the identified DNA into the host.
  3. Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

Tools of Recombinant DNA Technology

Restriction Enzymes

In 1963, two enzymes were discovered from Escherichia coli which restrict the growth of bacteriophage in it. One of these added methyl groups to DNA. The other cut the phage DNA (restriction endonuclease). The first restriction endonuclease discovered is Hind II. Hind II always cuts the DNA molecule at a particular point by recognizing a specific sequence of six base pairs. This is called the recognition sequence for Hind II. To date, around 900 restriction enzymes are isolated from 200 strains of bacteria, each of which recognizes different recognition sequences.

Restriction enzymes belong to nucleases. There are two kinds of nucleases:

  1. Exonuclease: Removes nucleotides from the free ends of the DNA.
  2. Endonuclease: Makes cuts at specific positions within the DNA.

Each restriction endonuclease recognizes a specific palindromic nucleotide sequence in the DNA. Palindromes are groups of letters that read the same both forward and backward, e.g., “MALAYALAM”. The palindrome in DNA is a sequence of base pairs that reads the same on the two strands when the orientation of reading is kept the same.

The restriction enzyme cuts the strand of DNA a little away from the center of the palindrome sites, but between the same two bases on the opposite strand. This leaves single-stranded portions at the ends. There are overhanging stretches called sticky ends on each strand. This stickiness of the ends facilitates the action of the enzyme DNA ligases. The foreign DNA and the host DNA are cut by the same restriction endonuclease, the resultant DNA fragments have the same kind of ‘sticky-ends’ and these can be joined together using DNA ligases.

Convention for naming restriction endonucleases: The first letter of the name comes from the genus. The second two letters come from the species of the prokaryotic cell from which the enzyme is isolated. The fourth letter is in capital form derived from the strain of microbes. The Roman letter followed is the order of discovery. Best example: EcoRI comes from Escherichia coli RY13.

Separation and Isolation of DNA Fragments

The cutting of DNA by restriction endonucleases results in the fragments of DNA. These fragments are separated by a technique called gel electrophoresis. Since the DNA fragments are negatively charged, they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. The most commonly used matrix is agarose, a natural polymer extracted from seaweed. 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. The separated fragments are visualized by staining them with ethidium bromide followed by exposure to UV radiation. The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is called elution.

Cloning Vectors

The plasmid and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA. Alien DNA linked with the vector multiplies its number equal to the copy number of the plasmid or bacteriophage.

Features of cloning vector:

  1. Origin of replication: This is the sequence where the replication starts called the origin. The alien DNA linked with the vector also replicates. It controls the copy number of the linked DNA.
  2. Selectable marker: It is required to identify recombinant from non-recombinant. Helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants. Transformation is a procedure through which a piece of foreign DNA is introduced into a host bacterium. Normally, the gene coding resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, or kanamycin sets are considered useful selectable markers for E. coli. The normal E. coli cells do not carry resistance against any of the antibiotics.
  3. Cloning sites: In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites (palindromic site) for the commonly used restriction endonucleases. Commonly used vector is pBR322 for E. coli. The ligation of foreign DNA is carried out at a restriction site present in one of the two antibiotic resistance genes. If a foreign DNA is ligated or inserted at the BamHI site of the tetracycline resistance gene in the vector pBR322, the recombinant plasmid will lose tetracycline resistance (insertional inactivation). The recombinant can be identified from the non-recombinant in the following steps:
    • All are grown in ampicillin medium.
    • One replica of the above plate is grown in ampicillin medium (control).
    • Another replica is grown in the medium containing both tetracycline and ampicillin. The colonies growing in plate-I but failing to grow in plate-II are identified as recombinants.

Alternative selectable marker: In E. coli, a plasmid called pUC-18 is used as a selectable marker, which is better than pBR322. The foreign DNA is introduced within the coding sequence of an enzyme β-galactosidase, which converts X-Gal (chromatogenic substrate) into galactose and 5-bromo-4-chloro-indigo (blue color). The non-recombinant produce enzyme and give blue-colored colonies. The recombinant is unable to produce β-galactosidase and does not produce blue-colored colonies after the addition of chromatogenic substrate i.e., X-Gal. This inactivation of insertion of foreign DNA is called insertional inactivation.

Vectors for Cloning Genes in Plants and Animals

Agrobacterium tumefaciens, a pathogenic bacterium of several dicot plants. This bacterium contains a plasmid called Ti-plasmid (tumor-inducing). In natural condition, A. tumefaciens transfers the T-DNA into the plant which transforms normal plant cells into a tumor and directs these tumor cells to produce the chemical required by the pathogen. Retroviruses in animals have the ability to transform normal cells into cancerous cells. The disarmed retroviruses are being used to transfer genes into animals. In Ti-plasmid the T-DNA is replaced by the gene of interest; still, A. tumefaciens is able to transfer the gene into the plant without causing tumors in plants.

Competent Host (for Transformation with Recombinant DNA)

DNA is a hydrophilic molecule; it 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. The bacterial cell is treated with divalent cations such as calcium, which increases the efficiency of DNA uptake by the bacteria. Recombinant DNA and the bacterial cells are incubated in ice, followed by placing them briefly at 42°C (heat shock) and then putting them back in ice. By microinjection, recombinant DNA is directly injected into the nucleus of the animal cell. Plant 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 disarmed pathogen vectors, when allowed to infect the cell, transfer the recombinant DNA into the host.

Process of Recombinant Technology

  1. Isolation of DNA.
  2. Fragmentation of DNA by restriction endonuclease.
  3. Isolation of desired DNA fragment by gel electrophoresis.
  4. Ligation of DNA fragment with a vector by DNA ligase.
  5. Transferring the recombinant DNA into the host.
  6. Culturing the host cells in a medium at a large scale in a bioreactor.
  7. Extraction of desired product by downstream processing.

Isolation of the Genetic Material (DNA)

Bacterial cell wall digested by lysozyme. Plant cell wall is digested by cellulase and pectinase. Fungal cell wall is digested by chitinase. RNA of the cellular content is digested by ribonuclease. Proteins are removed by proteases. Purified DNA is ultimately precipitated out after the addition of chilled ethanol. The precipitated DNA is separated and removed by spooling.

Amplification of Gene of Interest using PCR

PCR stands for Polymerase Chain Reaction: Multiple copies of the gene of interest can be synthesized in vitro. PCR includes the following steps:

  1. Denaturation: Double-stranded DNA is made single-stranded. It is done by heating the DNA at 94°C. Each single-stranded DNA is called the template strand.
  2. Annealing: Two sets of primers (small oligonucleotide chains that are complementary to the DNA at the 3′ end of the DNA template) are added to the medium. This is done at around 50°C.
  3. Extension: Deoxyribonucleotide triphosphates are added to the medium. Taq polymerase catalyzes the polymerization reaction using nucleotides, extending from the primer towards the 5′ end of the template. Taq polymerase is a heat-stable enzyme isolated from the bacterium Thermus aquaticus. It catalyzes the polymerization reaction at 74°C.

Obtaining the Foreign Gene Product or Recombinant Product

The protein-encoding gene is expressed in a heterogeneous host and is called a recombinant protein. The host is cultured in a continuous culture system provided in a bioreactor. A bioreactor provides optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen). The bioreactor converts the raw materials into a specific product or enzyme.

Downstream Processing

After biosynthesis inside the bioreactor, the product has to be subjected to a series of processes before it is ready for marketing. The process includes separation and purification, which are collectively referred to as downstream processing. The product has to be formulated with suitable preservatives. Such formulations must undergo clinical trials in the case of drugs.