Biotechnology and Recombinant DNA Technology: Techniques and Applications

Posted on Nov 28, 2024 in Biology

Biotechnology

For thousands of years, humankind has used microbial fermentation processes to obtain useful products. Modern biotechnology implies the deliberate manipulation of the genetic material of living organisms to make or modify a product, improve animals or plants, or develop microorganisms with specific skills for specific uses. The process of modern biotechnology requires understanding the mechanisms governing the decoding and expression of the information contained in genes and the development of specific tools and techniques, e.g., recombinant DNA technology, genetically engineered cell-cloning techniques, cell culture techniques, and tissue culture.

Recombinant DNA Technology

Recombinant DNA technology comprises a series of techniques that allow the manipulation of DNA, i.e., cutting, pasting, reproducing, and isolating specific DNA fragments from any organism. It also includes techniques to add a DNA snippet from a donor organism into another DNA molecule, called a vector, resulting in a DNA molecule with a new combination. Recombinant DNA is any DNA molecule formed by the union of segments of different origins.

Cellular Enzymes

In living cells, DNA is repeatedly cut and rejoined. Scientists have identified and purified specific types of enzymes for laboratory use:

  • Restriction enzymes or restriction endonucleases are synthesized by bacteria to protect their DNA from invaders. They function as chemical scissors, cutting foreign DNA into fragments.
  • Ligases are enzymes that link different DNA fragments with sticky ends.

DNA Fragment Analysis

Once restriction enzymes cut a piece of DNA, the resulting fragments can be separated and analyzed by different techniques, such as agarose gel electrophoresis. This technique separates DNA fragments according to their size and electrical charge. Agarose, a polysaccharide similar to gelatin, is used as a support medium through which the DNA can move. The procedure for agarose gel electrophoresis is as follows:

  1. A thin layer of agarose jelly is prepared in a mold, allowing the formation of small wells for samples.
  2. The gel is placed in an electrophoresis chamber containing a solution of water and salts, with electrodes attached to each end.
  3. Each well of the gel is loaded with a different sample using a micropipette. The samples contain mixtures of DNA fragments of different sizes to be separated.
  4. Since DNA is negatively charged, when an electric current is applied, the fragments move through the gel towards the positive electrode. Smaller fragments move faster and more easily through the gel mesh.
  5. After a period, the power is interrupted. To visualize the DNA bands, the agarose sheet is stained with ethidium bromide, which binds to DNA and fluoresces under ultraviolet light.

Agarose gel electrophoresis separates a mixture of DNA molecules into bands, each made up of thousands of DNA molecules of the same length. This technique allows obtaining a gene fingerprint (a characteristic and unique banding pattern of an organism’s DNA). The gene fingerprint is the DNA fingerprint and makes it possible to ascertain the identity of an individual, e.g., in crime investigations, paternity testing, and ancient mysteries.

Hybridization Using DNA Probes

Hybridization is the process where two single strands of DNA with complementary base sequences bind together to form a double-stranded DNA molecule. It is a fundamental technique in biotechnology to identify the presence of a gene encoding a protein of interest on a chromosome. This is achieved through a hybridization test with a DNA probe. A DNA probe is an artificial single-stranded DNA fragment labeled with radioactivity or fluorescence, with a nucleotide sequence complementary to the gene sequence to be detected. Thousands of genes can be analyzed simultaneously using DNA chips or biochips. Biochips are glass slides with tiny amounts of single-stranded DNA fixed in microscopic cells. Each cell acts as a probe for a particular gene. Thousands of microscopic cells are placed in a grid on the glass sheet, allowing for thousands of parallel hybridization reactions. Since the location of each DNA probe is known, any hybrid can be identified. Biochip technology is used to:

  • Detect mutations
  • Control gene expression
  • Diagnose infectious diseases
  • Personalize drug treatment and suggest new therapies

DNA Cloning

Cloning signifies the production of genetically identical individuals by asexual reproduction. Cloning of a DNA fragment is the creation of millions of identical copies of that fragment. To clone a DNA fragment, it must be inserted into a cloning vector, a small DNA molecule capable of entering a bacterium and replicating inside it. Bacterial plasmids are commonly used as vectors. The cloning method is as follows:

  1. The plasmid DNA and the DNA fragment to be cloned are cut with the same restriction enzyme. The recombinant plasmids (plasmids with inserted DNA fragments) are incubated with a bacterial culture under conditions that allow only bacteria with a recombinant plasmid to incorporate it. This process is called transformation.
  2. Since the plasmid carries a gene conferring antibiotic resistance, transformed bacteria can be selected by growing them on plates containing the antibiotic. Only bacteria with the recombinant plasmid will survive; others will die.
  3. Each colony of transformed bacteria is isolated and grown. As the bacteria multiply, so do the plasmids and the inserted DNA fragments.

The collection of DNA clones from a DNA of interest is called a DNA library or genomic library.

Polymerase Chain Reaction (PCR) DNA Amplification

DNA can be amplified inside a test tube without cloning. This technique is based on the polymerase chain reaction (PCR). PCR is a chain reaction that generates millions of copies of a specific DNA segment through multiple cycles of DNA replication in vitro. The starting materials are the DNA sample to be amplified, the four nucleotides, two short single-stranded DNA molecules (primers), and a heat-resistant DNA polymerase. PCR is a tool used to amplify DNA segments from a wide variety of sources, for example, to make a prenatal diagnosis of genetic diseases.

DNA Sequencing

The determination of the nucleotide sequence is a fundamental part of any information about a DNA fragment. Automated and computerized techniques allow for easy and fast sequencing.