recombinant dna

3.6 - Recombinant DNA

Biotechnology

  • Definition: Use of a NATURAL BIOLOGICAL SYSTEM to produce a product or desired end, such as hormones, antibiotics, drugs, etc.

  • Advances in Biotechnology: Progress accelerated notably with the capability to produce recombinant DNA.

Recombinant DNA (rDNA)

  • Definition: A form of DNA where DNA from ONE SPECIES (e.g., human insulin gene) is INSERTED into a SECOND SPECIES.

  • Vector Requirement: Recombinant DNA requires the use of a vector—a tool or agent that can transport DNA from one species into another’s DNA, allowing the introduction of foreign DNA.

Types of Vectors

  1. Plasmids

    • Description: Circular DNA that can REPLICATE INDEPENDENTLY of chromosomes.

    • Source: Found in some bacteria.

  2. Viruses

    • Description: Biologic elements that can infect other organisms and transfer genetic material.

Restriction Enzymes

  • Definition: Proteins produced by bacteria that CUT DNA at specific locations.

  • Cleavage Mechanism: Each restriction enzyme cleaves DNA at a RESTRICTION SITE, which is a specific sequence of nucleotides ranging from 4 to 9 base pairs in length.

    • Example: EcoR1 restriction enzyme recognizes the sequence GAATTC.

Functionality of Restriction Enzymes

  • Restriction enzymes continuously scan a DNA molecule and make cuts whenever they find their specific nucleotide sequence.

  • EcoR1 Specificity: Cuts between G and A of the recognized sequence in each DNA strand.

    Destruction of Base Pair Bonds

    • After the cuts are made, the DNA is held together primarily by WEAK HYDROGEN BONDS between the bases.

    • These bonds are easy to break.

  • Sticky Ends: The cut sites do not align directly across from each other, resulting in “sticky ends” which facilitate bonding with complementary DNA.

Recombinant DNA Process

  1. Gene Extraction: Restriction enzymes cut out the gene of interest (e.g., insulin gene from human DNA).

  2. Plasmid Preparation: Plasmids are isolated from bacterial cells and cut with the same restriction enzymes to enable the insertion of foreign DNA.

  3. Formation of rDNA: The gene of interest and the plasmid are combined, and DNA ligase seals them together, forming recombinant DNA.

  4. Host Cell Transferral: The rDNA is introduced into a host bacterium, which incorporates the gene.

  5. Cloning: The plasmid reproduces during cell division. If the gene expresses correctly, the host bacteria will produce the target protein (e.g., insulin).

Biotechnology Applications

Protein Production via Recombinant DNA

  • Can create therapeutic products like insulin, human growth hormone, and hepatitis B vaccine in transgenic bacteria.

GMOs (Genetically Modified Organisms)

  • Definition: Organisms with foreign DNA or genes inserted into them.

    • Such organisms are termed TRANSGENIC ORGANISMS.

    • Transgenic bacteria, plants, and animals are classified as GMOs, and the products they yield are referred to as biotechnology products.

  • Examples:

    • GMO Bacteria: Engineered to resist ice crystal formation, incorporated into strawberry plants to create frost-resistant berries, thus enhancing product yield.

    • Oil-eating bacteria: Enhanced for environmental clean-up efforts post-oil spills.

    • GMO Animals: Foreign genes inserted into animal eggs to produce target products in their milk, used for therapies related to human growth hormone, cystic fibrosis treatments, and cancer drugs.

    • GMO Plants: Examples include Pomatoes (combining traits of potatoes and tomatoes). Crops such as corn, cotton, and potatoes are modified for pest resistance.

Gene Therapy

  • Definition: The insertion of genetic material into human cells for treating genetic disorders and illnesses, such as cystic fibrosis, Alzheimer's, some cancers, and AIDS.

  • Mechanism: Modified viruses introduce normal (non-mutated) human genes into the body.

  • Issues in Gene Therapy:

    • High costs: Treatment is very expensive.

    • Complexity: Difficulties in manipulating human genes lead to potential unknown side effects, including the risk of developing leukemia in some patients.

    • Targeting: Challenges in inserting genes at the correct genomic location.

    • Ethical concerns: Experimentation on humans raises ethical questions.

    • Eugenics: It raises the prospect of engineering “designer babies”.

Vaccine Production

  • Examples of Vaccines: Hepatitis B, HIV, flu vaccines produced using genetic technologies.

  • Advantages: Safer than traditional vaccines, producing immune responses without the complications associated with conventional vaccines.

CRISPR Technology

  • Definition: A groundbreaking technology allowing scientists to edit DNA at specific locations in the genome. Initially discovered as a bacterial defense mechanism against viruses.

  • Adaptation: Scientists have adapted this system to cut and modify genes in living cells.

Components of CRISPR

  1. Guide RNA (gRNA):

    • Short RNA sequence that matches the DNA target.

    • Directs the CRISPR system to the correct genomic location.

  2. Cas9 Enzyme:

    • Functions as molecular scissors, cutting DNA at designated sites.

CRISPR Process

  • The guide RNA leads Cas9 to the target DNA location. Once at the site, Cas9 facilitates the DNA cut.

  • The cell repairs the cut; during this process:

    • Genes can be disabled.

    • New DNA can be inserted.

    • Mutations can be corrected.

Applications of CRISPR

  • Study gene function.

  • Treat genetic diseases.

  • Improve crops.

  • Develop innovative medical therapies.

DNA Fingerprinting

  • Process: Involves the extraction of DNA, digestion using restriction enzymes (REN), probing, and electrophoresis to visualize the DNA.

  • Application Example: In crime scene investigations, comparing DNA samples from suspects and crime scene evidence (e.g., blood, hair, skin cells).