Recombinant DNA Technology, Cloning and PCR

Recombinant DNA Technology, Cloning, and PCR

Learning Outcomes

• Recombinant DNA: Scientists utilize knowledge of gene structure and regulation to express modified genes.

  • 17A: Describe how scientists can use various tools and techniques to make recombinant DNA.

  • Tools include enzymes, vectors, and host cells.

  • Techniques include gel electrophoresis, sequencing, and PCR.

  • 17B: Describe gene cloning and construct a flow chart for designing recombinant DNA that expresses a particular type of protein through gene cloning.

  • 17C: Outline a specific example of a human disease or societal problem for which recombinant DNA technology provides a solution.

What is Cloning?

• Reproductive Cloning:

  • Creating an identical copy of an organism, ensuring they are genetically identical.

• Molecular (Gene) Cloning:

  • Highlighting the copying of specific genes rather than whole organisms.

Reproductive Cloning

• Genomic Equivalence:

  • Nearly all cells in an organism have genomic equivalence, meaning they contain the same genes.

  • Mechanism:

  • Reproductive cloning uses one or more somatic cells from a multicellular organism to create another genetically identical individual.

Nuclear Transplantation in Animals

• Process:

  • The nucleus of an unfertilized egg cell or zygote is replaced with the nucleus from a differentiated cell.

• Notable Example:

  • In 1997, Scottish researchers successfully cloned a lamb named Dolly from an adult sheep using nuclear transplantation.

Cloning Process Steps

• Nucleus Removal:

  • Nucleus from a mammary cell donor is extracted.

  • Egg cell donor's egg cell is used.

  • Cultured mammary cells are semi-starved to arrest the cell cycle and induce dedifferentiation.

• Fusing Cells:

  • The nucleus from the mammary cell is introduced to the egg cell.

• Development in Uterus:

  • The resulting embryo is implanted into the uterus of a surrogate mother, leading to embryonic development of the cloned lamb, Dolly.

  • Result:

    • The cloned animal is genetically identical to the mammary cell donor but is different from the egg cell donor and surrogate mother.

Advances Since Dolly

• Ordinary cells can be reprogrammed into induced pluripotent stem cells (iPS).

• Gene-edited monkeys were cloned in China in 2019.

• The first embryonic editing using CRISPR-Cas9 was performed resulting in babies named Lulu and Nana.

Molecular Cloning

• Definition:

  • A method to create multiple copies of a gene, also called gene cloning.

  • Requires origin of replication (ORI) sequences and promoter sequences for gene expression.

Components of Molecular Cloning

• Necessary Components:

  • DNA to be cloned

  • Vectors:

  • Carry DNA inside cells and replicate.

  • Types include plasmids, bacteriophages, or viruses, and artificial chromosomes.

  • Bacteria

  • Enzymes

Cloning Vectors

• Plasmids:

  • Origin: Bacteria.

  • They are circular DNA molecules distinct from the bacterial chromosome (extrachromosomal).

  • They are capable of replication and contain origins of replication sequences.

Enzymes in Molecular Cloning

• Bacterial Restriction Enzymes (endonucleases):

  • Cut DNA at specific sequences called restriction sites, resulting in restriction fragments.

• DNA Ligase:

  • An enzyme that seals the nicks in the DNA backbone after fragments have been joined.

Restriction Endonucleases Example

• Sequence Example:

  • 5'-C-C-A-G-G-A-A-T-T-C-G-A-T-G-G-3'

  • 3'-G-G-T-C-C-T-T-A-A-G-C-T-A-C-C-5'

Animation of Restriction Enzymes

• Features a demonstration of how different restriction enzymes interact with DNA sequences.

Making a Recombinant DNA Using Restriction Enzymes and DNA Ligase

• Process:

  • A restriction enzyme cuts the sugar-phosphate backbones of the DNA, creating sticky ends.

  • A DNA fragment from another source is added, allowing base pairing via the sticky ends.

  • DNA ligase is then used to seal the strands, forming recombinant DNA.

• Example Combination:

  • Sequence illustration showing potential combinations of DNA from different sources.

Gene Cloning and Its Uses

• Bacterial Recombinant DNA:

  • Gene inserted into a plasmid (which serves as a cloning vector).

  • Resulting recombinant bacterium contains the gene of interest.

  • Host cells are grown in culture to clone the cells containing the gene. - Applications:

  • Producing human growth hormone to treat stunted growth.

  • Creating plants with pest resistance.

  • Altering bacteria for bioremediation of toxic waste.

  • Developing proteins that dissolve blood clots in heart attack therapy.

Medically Useful Products of Biotechnology

• List of Products and Their Uses:

  • Colony-stimulating factor: Stimulates white blood cell production in cancer/AIDS patients.

  • Erythropoietin: Prevents anemia in patients undergoing kidney dialysis.

  • Factor VIII: Replaces clotting factor in hemophilia A patients.

  • Growth hormone: Replaces missing hormone in people of short stature.

  • Insulin: Stimulates glucose uptake in Type I diabetes patients.

  • Platelet-derived growth factor: Stimulates wound healing.

  • Tissue plasminogen activator: Dissolves blood clots in heart attacks and strokes.

  • Vaccine proteins: Used to prevent and treat various infectious diseases (e.g., Hepatitis B, Herpes, Influenza, etc.).

Recombinant Therapeutic Proteins: Insulin

• Context:

  • Type 1 Diabetes: Characterized by the excretion of large amounts of glucose in the urine due to the body's inability to make insulin.

  • Insulin is produced in the pancreas and modified in the endoplasmic reticulum and Golgi apparatus.

• Types of Diabetes:

  • Type 1 Diabetes: Insulin not made.

  • Type 2 Diabetes: Insulin resistance.

Process of Insulin Gene Expression

• In Cells:

  • Transcription occurs, followed by post-transcription processing.

  • Translation leads to the formation of preproinsulin, which is processed into proinsulin and finally secreted in response to a signal.

Diagram of Gene Expression Process

• Shows enhancer elements, control elements, poly-A signal sequences, termination regions, upstream and downstream DNA elements, and processing of RNA transcripts.

Polymerase Chain Reaction (PCR)

• Definition:

  • A method for amplifying DNA in vitro.

• Inventor:

  • Dr. Kary Mullis (1944-2019)

• Process:

  • Denatures genomic DNA, becomes single-stranded. - Uses temperature cycles for elongation and annealing of primers with DNA polymerase (specifically Taq polymerase).

PCR Temperature Cycling Steps

1. Initial Denaturation: 94°C for 5 minutes.

2. Annealing: 50-60°C for 2 minutes where primers base-pair with target sequences.

3. Extension: 72°C for 2-5 minutes to elongate DNA strands.

PCR Output Example

• Illustrates exponential DNA amplification, moving from 2 copies of DNA to 64 copies after several cycles.

Learning Objectives Overview

• Explain the process of nuclear transplantation used to create Dolly.

• Differentiate between cloning vectors and expression vectors.

• Detail each step and enzyme utilized in molecular cloning.

• Discuss the challenges of expressing eukaryotic genes in prokaryotic systems.

• Describe the principles of PCR and how it compares to DNA replication.

Vocabulary

• Reproductive Cloning

• Molecular Cloning

• Nuclear Transplantation

• Restriction Enzymes/Endonucleases

• Reverse Transcription

• Reverse Transcriptase

• Vector

• Plasmid

• Recombinant DNA

• Restriction Sites

• Restriction Fragments

• Polymerase Chain Reaction (PCR)

• Denaturation, Annealing, Extension

Recommended Reading

• Concept 16.2: Cloning of organisms demonstrated that differentiated cells can be reprogrammed, leading to stem cell production.

• Concept 13.3: Understanding DNA structure and replication is foundational for genetic engineering.

• Focus on amplifying DNA through PCR and its application in cloning.