Comprehensive Notes on DNA Cloning

DNA Cloning

Manipulating the Genome

  • Recombinant DNA: DNA in which nucleotide sequences from two different sources are combined in vitro into the same DNA molecule.
  • Genetic Engineering: Direct manipulation of genes for practical purposes (e.g., CRISPR gene editing).
  • Biotechnology: Manipulation of organisms or their components to make useful products.
    • Examples: Pest resistance in crops, bacteria to clear toxic waste, proteins that dissolve blood clots, and human growth hormone.

Genetic Engineering Technique

  • Introduction of manipulated genetic material into a cell in such a way as to replicate and be passed on to progeny cells.

Gene Cloning

  • Insertion of a DNA fragment carrying a gene into a cloning vector, followed by propagation of recombinant DNA molecules to produce many copies.
  • This process is also known as gene manipulation.

Basic Steps of Gene Cloning

  1. Construction of recombinant DNA molecule.
  2. Transport of the recombinant DNA to the host cell.
  3. Multiplication of recombinant DNA molecule.
  4. Division of the host cell.
  5. Numerous cell divisions resulting in a clone.

Gene Cloning Technique

  • Process Overview:
    1. Gene of interest is inserted into a plasmid to create Recombinant DNA (plasmid).
    2. Plasmid is put into bacterial cell.
    3. Recombinant bacterium contains DNA of chromosome (“foreign” DNA).
    4. Host cell is grown in culture to form a clone of cells containing the cloned gene of interest.
    5. Protein is expressed from gene of interest and harvested or copies of gene are obtained.
  • Applications:
    • Basic research on gene and protein.
    • Gene for pest resistance inserted into plants.
    • Gene used to alter bacteria for cleaning up toxic waste.
    • Protein dissolves blood clots in heart attack therapy.
    • Human growth hormone treats stunted growth.

Cloning Process

  1. Gene of interest is cut out with a restriction enzyme.
  2. Host plasmid is cut with the same restriction enzyme.
  3. Gene is inserted into the plasmid and ligated with ligase.
  4. New plasmid is inserted into a bacterium (transformation).

Gene Cloning Tools and Techniques

  • Vehicles:
    • Transport the gene into the host cell and are responsible for its replication.
    • Must be capable of entering a host cell and replicating to produce multiple copies of itself.
  • Vector:
    • A DNA molecule capable of replication in a host organism.
    • A gene is inserted into it to construct a recombinant DNA molecule.

Cloning Vector Requirements

  1. Must contain an origin of replication recognized in the host cell for replication along with the carried DNA.
  2. Should carry selectable markers (traits that enable cells containing the vector to be selected or identified).
  3. Needs a single cleavage site for each of one or more restriction enzymes used.

Characteristics of a Vector

  • It must be able to replicate.
  • There must be some way to introduce vector DNA into a cell.
  • There must be some means of detecting its presence.
  • Examples:
    • Plasmids
    • Bacteriophage-based cloning vectors
    • Yeast artificial chromosomes (YACs)
    • Bacterial artificial chromosomes (BACs)
    • Mammalian artificial chromosomes (MACs)

Plasmid Characteristics

  • Small (3-5 kb of DNA).
  • Contain suitable markers (antibiotic resistance).
  • Contain suitable restriction sites for inserting DNA fragments for cloning.

Plasmid Cloning Strategy

  1. Enzyme restriction digest of DNA sample.
  2. Enzyme restriction digest of DNA plasmid vector.
  3. Ligation of DNA sample products and plasmid vector.
  4. Transformation with the ligation products.
  5. Growth on agar plates with selection for antibiotic resistance.

Plasmid Cloning

  • The plasmid and the foreign DNA are cut by the same restriction enzyme (e.g., EcoRI).
  • When mixed, the sticky ends anneal, joining the foreign DNA and plasmid.
  • Nicks in the sugar-phosphate bonds are sealed by DNA ligase.

Selection of Recombinant Plasmids

  • Foreign DNA is inserted into the middle of the lacZ gene.
  • Bacteria are transformed by the plasmids.
  • Bacteria with an intact plasmid produce ββ-galactosidase, which cleaves X-gal and makes the colonies blue.
  • Bacteria with a recombinant plasmid do not synthesize ββ-galactosidase; their colonies remain white.
  • Bacteria without a plasmid will not grow on medium with ampicillin and X-gal.

Types of Cloning

  1. Recombinant DNA technology or DNA cloning.
  2. Reproductive cloning.
  3. Therapeutic cloning.

Recombinant DNA Technology (DNA Cloning)

  • Terms “recombinant DNA technology”, “DNA cloning”, “molecular cloning” or “gene cloning” all refer to the same process: the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid.
  • The DNA of interest can then be propagated in a foreign host cell.
  • This technology has been around since the 1970’s, and it has become a common practice in molecular biology labs today.

Gene Cloning with Different Vectors

  • Vectors:
    • Plasmid.
    • Virus.
  • Process:
    • Recombinant DNA molecule is transformed into a host cell (bacterium or virus).
    • Cell division or cell lysis occurs, resulting in copies of the foreign gene.

Application: Using Plasmids to Produce Insulin

  • Human insulin-producing gene is extracted from a human pancreas cell.
  • Plasmid DNA is cut with restriction enzymes.
  • Recombinant DNA is created by inserting the human insulin-producing gene into the plasmid.
  • Recombinant DNA is introduced into a bacterial cell.
  • Recombinant bacteria multiply and produce human insulin in a fermentation tank.
  • Human insulin is extracted and purified.

How Insulin Is Made from Recombinant DNA

  • Recombinant bacterium produce insulin in fermentation tank.
  • Insulin is harvested

Reproductive Cloning

  • Dolly was created by reproductive cloning technology, in a process called “Somatic Cell Nuclear Transfer” (SCNT).
  • Scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus has been removed.
  • The reconstructed egg is treated with chemicals or electric current to stimulate cell division.
  • Once the clone embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth.

Reproductive Cloning Process

  • Process:
    • Remove DNA from unfertilized egg.
    • Remove udder cell/skin cell from animal to be cloned.
    • Fuse cells.
    • Early embryo with donor DNA.
    • Implant in surrogate.

Cloning Techniques

  • Nuclear Transfer:

    1. Extract DNA from oocyte (egg).
    2. Extract DNA from donor cell in G0 stage of cycle.
    3. Inject DNA into empty oocyte and fuse with electricity.
    4. After a few division in culture, implant in surrogate mother.

  • Microinjection Technique

Reproductive Cloning

  • Tissue cell donor with cells maintained in the lab so they do not grow or divide, Nucleus is removed.
  • Donor supplies unfertilised eggs. Nucleus is removed
  • Nucleus fuses with empty egg after electric current applied
  • The reconstructed embryo grows for 7 days.
  • Embryo's implanted into surrogate mother.
  • Cloned animal is born with exact DNA as the tissue cell donor.

Fertilization vs. Cloning

  • Fertilization:
    • Egg cell undergoes meiosis.
    • Sperm cell undergoes meiosis.
    • Fertilization occurs, resulting in a fertilized embryo with combined genetics of parents.
  • Cloning:
    • Remove egg nuclear material.
    • Isolate donor cell nuclear material.
    • Insert donor nuclear material into egg cell.
    • Results in a cloned embryo genetically identical to donor

Technical Hurdles in Cloning

  • Complications, low success rates (e.g., Dolly was the result of 277 attempts).
  • Developmental problems (lungs, immune system, weight gain).
  • Cell cycle coordination issues.
  • Reprogramming process challenges.
  • Methylation disturbances.
  • Potential improper regulation of Oct4 gene.

Therapeutic Cloning

  • Production of human embryos for research.
  • Goal is to harvest stem cells to study human development and treat disease.
  • Stem cells can generate virtually any type of specialized cell in the human body.

Therapeutic Cloning Process

  1. Extract DNA from adult cells.
  2. Create cloned embryos.
  3. Extract embryonic stem cells.
  4. Destroy embryo.

Potential Applications of Therapeutic Cloning

  • Direct tissue engineering of spare parts on demand.
  • Genetically identical to recipient (no rejection).
  • Grow whole organs to alleviate organ shortage.
  • Grow non-regenerating cells (brain cells for Parkinson’s, pancreatic islet cells for diabetes).

Gene Therapy

  • Needle injects replacement gene to rejuvenate pigment layer in treated area.

Gene Therapy Mechanism

  1. Cell Binding Factor
  2. Regulatory Promotor Region
  3. Nucleus
  4. Cytoplasm
  5. Cell Death

How Cloning Technologies Can Be Used

  • Gene therapy for genetic conditions using virus vectors to carry corrected genes.
  • Genetic engineering of food crops for improved taste, nutritional value, or disease resistance.
  • Reproductive cloning to repopulate endangered or difficult-to-breed animals.

Cloning Organs for Transplants

  • Therapeutic cloning to generate tissues and organs for transplants.
  • Stem cells used to generate an organ that is a genetic match to the recipient.
  • Transplantation without risk of tissue rejection.

Risks of Cloning

  • Expensive and inefficient.
  • High failure rate in producing viable offspring (more than 90% fail).
  • Compromised immune function and higher rates of infection, tumor growth, and other disorders in cloned animals.