DNA Technology Overview and Cloning Techniques

DNA Technology Overview

• DNA technology manipulates genes and organisms for practical purposes.
• Traditional techniques include breeding plants and animals, and using microorganisms to make products like bread, wine, and cheese.
• Modern biotechnology involves genetic engineering and therapeutic cloning.

DNA Cloning

• DNA cloning produces identical copies of a gene or DNA segment.
• Genetic engineering involves moving genes between organisms.
• The universality of molecular genetics allows genes to be moved and manipulated.
• DNA cloning has diverse biotechnological applications.

Recombinant DNA Engineering

• Recombinant DNA is DNA assembled in an order not found in nature.
• Plasmids are small circles of DNA retained by bacterial cells.
• Restriction enzymes recognize and cut specific DNA sequences.
• DNA ligase seals DNA strands.

Simple Cut-Paste Steps for Cloning

• Restriction enzymes cut DNA.
• DNA fragments are mixed with compatible sticky ends.
• DNA ligase forms bonds to create a continuous DNA helix.

Isolating a Unique Clone

• Induce bacteria to take up the plasmid.
• Selection involves using antibiotic resistance genes to identify transformed bacteria.
• All descendent bacteria now carry recombinant DNA in the plasmid.

PCR for DNA Copies

• PCR (Polymerase Chain Reaction) amplifies DNA copies.
• It doubles each cycle, making huge numbers of copies.
• PCR is useful for cloning and diagnostics.
• Primers define the target DNA region to amplify.

PCR Cycle Steps

• Denaturation: Separates DNA strands by heating.
• Annealing: Allows primers to bind to single strands.
• Extension: DNA polymerase extends primers, doubling the region between primers in each cycle.

Applications of DNA Technology

• Forensic applications: Unique differences (polymorphisms) in Short Tandem Repeats (STRs) are used for DNA forensics. For example, Earl Washington's conviction.
• Biopharmaceutical applications: Techniques amplify genes for useful proteins, clone those genes into plasmids, and express/purify proteins. Insulin treatment of diabetes, antibodies for cancer treatment, growth hormones for dwarfism, and tissue plasminogen activator for heart attack treatment are examples.
• Agricultural applications (Pharm animals and Transgenic crops): Animals can be modified to produce desirable proteins (e.g., anti-coagulants). Plants can be modified for pest resistance, herbicide tolerance, and improved nutrition.
• Regenerative medicine: Therapeutic cloning replenishes diseased tissues using healthy stem cells to enable regenerative medicine.

Plant Organismal Cloning

• Entire plants can be cloned from single cells.
• Mature plant cells can become totipotent, which allows them to grow all types of plant cells.
• Totipotency is the ability of a cell to differentiate into any cell type.

Animal Organismal Cloning

• Totipotency is more difficult to achieve in animal cells.
• Organismal cloning creates whole organisms genetically identical to a single cell.
• This involves nuclear transplantation (e.g., Dolly the sheep).

Dolly the Sheep (1997)

• Organismal cloning by nuclear transplantation.
• Low efficiency (<1%).
• Dolly had health problems and is not a routine technique.
• Cloning of other animals is possible but problematic.

Therapeutic Cloning and Stem Cells

• Therapeutic cloning replicates diseased tissues using healthy stem cells.
• Stem cells reproduce indefinitely and can differentiate into specialized cells.
• Pluripotency is the ability of a cell to become many cell types but not all cell types.

Sources of Human Stem Cells

• Embryonic stem cells: Easy to culture, can differentiate into many cell types but raise ethical issues relating to using early-stage human embryos.
• Adult stem cells: Very low abundance, can only differentiate into a limited number of cell types.

Induced Pluripotent Stem Cells (iPS cells)

• Adult cells can be reprogrammed to be pluripotent (iPS cells) using supplemental genes.
• These iPS cells have high potency and raise no ethical dilemma.
• They offer the potential for individualised medicine using a person's own cells.

Learning Outcomes for Lecture

• Understand the basis of gene cloning.
• Apply gene technology to gene expression and function.
• List real-world uses of DNA technology.
• Understand the general concept and steps of therapeutic and organismal cloning.
• Recognize different stem cell sources.