Understanding Cloning Processes and Applications

• Cloning is a process for producing genetically identical copies of a biological entity.
• A a copied material with the same genetic makeup as the original is called a clone.
• Genes, cells, tissues, and entire organisms like sheep have been cloned.
• Cloning is useful for making multiple copies of a specific sequence.

Applications of Cloned DNA

• Cloning enables detailed analysis and downstream applications.
• Helps in figuring out a gene's function.
• Cloning is useful in studying how mutations impact gene function.
• Genes characteristics like size, expression, and tissue distribution can be investigated.
• Cloning can be used to produce large concentration of the protein coded for the gene.

Types of Artificial Cloning

• Gene cloning produces copies of genes or segments of DNA.
• Reproductive cloning produces copies of whole animals.
• Therapeutic cloning produces embryonic stem cells for creating tissues to replace injured or diseased tissue.

Gene Cloning Mechanism

• Foreign DNA is inserted into the genetic material of a carrier called a vector.
• Vectors include bacteria, yeast cells, viruses, or plasmids.
• Plasmids are small, circular DNA molecules found in bacteria.
• After gene insertion, the vector multiplies under laboratory conditions, resulting in the gene being copied many times.

Steps of Cloning

• Preparation involves inserting foreign DNA (the desired sequence) into an acceptor vector (vehicle).
• Vectors can be linear or circular
• The vector and foreign DNA are cut using the same restriction enzymes.
• Insertion (Recombination/Cloning): Results in recombinant DNA, also known as a clone, hybrid, or chimera.
• Transformation (Transfection/Infection): Recombinant DNA is moved to a host cell like E. coli.
• Screening (Plating): Used to differentiate between transformed and non-transformed bacteria
• Screening: Used to discriminate between transformed bacteria with recombinant DNA, and those without.
• Bacteria can be transformed or non-transformed.
• Growing: Transformed recombinant bacteria are grown in a liquid medium followed by filtration
• Isolation: Involves separating the vector and the insert

General DNA Cloning Protocol

• Chosen DNA is cut from the source organism using restriction enzymes.
• The piece of DNA is pasted into a vector, with the ends of the DNA joined with the vector DNA by ligation.
• The vector is introduced into a host cell by transformation.
• Host cells copy the vector DNA along with their own DNA, creating multiple copies of the inserted DNA.
• Vector DNA is isolated or separated and purified from the host cell' DNA.
• Recombinant DNA technology is used to clone the DNA

Standard DNA Cloning Steps

• Choice of host organism and cloning vector Second Step: Vector DNA Preparation
• Preparation of the DNA to be cloned.
• Creation of recombinant DNA. Fifth Step: Introduction of recombinant DNA into a host organism.
• Selection (Isolation) of organisms containing recombinant DNA.
• Screening for clones with desired DNA inserts and biological properties.

Host Organism and Cloning Vector

• Host: Bacterium E. coli (Escherichia coli).
• Vector: Plasmid.
• E. coli and plasmid vectors are commonly used because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment.
• Typically, a vector contains four DNA segments: an origin of DNA replication, unique restriction endonuclease recognition sites, a selectable genetic marker gene, and an additional gene for screening cells containing foreign DNA.

Vector DNA Preparation

• The cloning vector is treated with a restriction endonuclease to cleave the DNA where foreign DNA will be inserted.
• Vector DNA and foreign DNA are cleaved using the same restriction enzymes.
• Most modern vectors have unique cleavage sites within the vector molecule for single-site cleavage.
• A cleavage vector may be treated with alkaline phosphatase to dephosphorylate the vector ends.
• Vector molecules with dephosphorylated ends cannot replicate unless foreign DNA integrated into the cleavage site restores replication.

Preparation of DNA to be Cloned

• DNA is extracted from tissue sources or extinct animals.
• Simpler methods purify DNA to remove contaminating proteins (phenol extraction), RNA (ribonuclease), and smaller molecules (precipitation or chromatography).
• PCR is used to amplify specific DNA or RNA (RT-PCR) sequences before molecular cloning.
• Purified DNA is treated with a restriction enzyme to create fragments capable of being linked to the vector.

Creation of Recombinant DNA

• DNA from the vector and foreign source are mixed at appropriate concentrations and exposed to DNA ligase to catalyze the ligation process.
• The resulting DNA mixture contains randomly joined ends and is ready for introduction into the host organism.

Introduction of Recombinant DNA into Host Organism

• The DNA mixture is introduced into a living cell or host.
• Four experimental methods are used:
  • Transformation: Microorganisms take up and replicate DNA from their local environment. -Transfection: Used in mammalian cell culture.
  • Transformation & Transfection: Requires cell preparation through a special regime and chemical treatment.
  • Electroporation: Uses high-voltage electrical pulses to translocate DNA across the cell membrane.
  • Transduction: Packages DNA into virus-derived particles and uses these virus-like particles to introduce the encapsulated DNA into the cell.
  • Electroporation & Transduction are specialized and efficient methods for moving DNA into cells.

Selection (Isolation) of Organisms Containing Recombinant DNA

• Only a small fraction of cells take up DNA.
• Only cells that possess the selectable marker gene encoded by the vector can actively replicate DNA.
• A selectable marker is usually a gene conferring resistance to an antibiotic.
• Cells with the plasmid survive when exposed to the antibiotic; those without plasmid sequences die.

Screening for Clones with Desired DNA Inserts and Biological Properties

• For screening clones containing recombinant DNA, a chromogenic substrate known as X-gal is added to the agar plate.
• B-galactosidase production hydrolyzes X-gal, making an insoluble blue pigment.
• Colonies formed by non-recombinant cells therefore appear blue, while the recombinant ones appear white.
• Desired recombinant colonies are picked and cultured.

Blue-White Screening System

• Is a screening technique for rapid detection of recombinant bacteria in vector-based molecular cloning experiments, using Ex: PUC19 vector.
• DNA is inserted into a host cell viable for transformation and grown in the presence of X-gal.
• Cells transformed with vectors containing recombinant DNA produce white colonies.
• Cells transformed with non-recombinant plasmids grow into blue colonies.
• Foreign DNA is inserted into a sequence that encodes part of beta-galactosidase (lacz), an enzyme whose activity results in formation of blue colored colony, resulting in enzymes activity being disabled, so that transformed DNA remain colorless.

Note

• The term DNA library commonly refers to the total population of individual clones that are created during a molecular cloning experiment.
• The collection of recombinant vectors containing cloned sequences that cover the entire genome of a specific organism is called a DNA Library.

Application of Molecular (DNA) Cloning

• Genome organization and gene expression:
  • Elucidation of the complete DNA sequence of the genome of various species. -Exploration of genetic diversity within individual species. -Molecular clones used to generate probes for examining gene expression.
• Cloned genes provide tools to examine biological function of individual gene importance such as making mutation of using regional mutagenesis

Production of Recombinant Proteins

• The molecular clone of a gene can lead to the development of organisms that produce the protein product, termed a recombinant protein.
• Producing an organism that produces an active protein can be difficult in desirable qualities.
• Recombinant proteins are medically useful and can correct a defective/poorly expressed gene.
• Some examples are recombinant factor VIII and recombinant insulin.
• Recombinant proteins aid in life-threatening emergencies like tissue plasminogen activator.
• Recombinant vaccines consist of subunit proteins to immunize people against infectious diseases.
• Recombinant proteins are standard material for diagnostic laboratory tests.

Transgenic Organisms

• Cloned genes can be inserted into organisms to create transgenic organisms, also known as GMOs.
• GMOs are used commercially to produce pharmaceuticals or other compounds in animals and plants (pharming).
• GMO application: Herbicide-resistant crop plants.
• GMO application: Fluorescent tropical fish (Glofish) for home entertainment.

Gene Therapy

• Involves supplying a functional gene to cells lacking that function to correct a genetic disorder.
• Gene therapy is divided into germ cell therapy which has to result in permanent change, and somatic cell therapy which targets specific tissues where the treated cells are returned to the patient, targeting treatment or removal.

Cloning Vectors

• Cloning vectors carry foreign DNA into a host cell, replicate within the cell (bacterial or yeast), and produce copies of themselves and the foreign DNA.
• Types of Cloning Vectors: -Plasmid: cloning limit 0.1-10 kb. -Phage (bacteriophage): linearized with cloning limit of 8-20 kb . -Cosmid: extra chromosomal circular with cloning limit of 35-50 kb. -Bacterial Artificial Chromosomes (BAC): limit: 75-300 kb. -Yeast Artificial Chromosomes (YAC): has limit: 100-1000 kb.

Properties of Plasmids

• Extra-chromosomal circular DNA molecule.
• Circular DNA is more stable than linear DNA.
• The host cell autonomously and replicated by itself.
• Has a multiple cloning site (MCS). -Has gene ability to screening and drug resistance.
• Its is made of a known sequence.

Plasmid PR322

• pBR322, a plasmid, was among the first E. coli cloning vectors used
• The p stands for plasmid, and BR stands for Bolivar and Rodriguez.
• There are ampicillin resistance (AmpR) and tetracycline resistance (TetR) genes present with one type of origin of replication, at 4.7 Kilo base pairs in length
• Screening depend on two Antibiotic Resistance ( 2 AB): AmpR gene is used to differentiate with bacteria and the Tetr gene is used to discriminate recombinant bacteria with DNA.

Twin Antibiotic Resistance Screening

• Screening is conducted by insertional inactivation of a resistance gene

Non-Transformed Bacteria

• Non- transformed means that it cannot grow on ampicillin agar plate

Transformed Bacteria

• Transformed means it grows in ampicillin medium because it has a Plasmid with AmpR gene

Transformed Bacteria with recombinant DNA

• The host organism can grow only in ampicillin medium and cannot grow on tetracycline medium or AmpR gene is used to discrimate between recombinant DNA
• Recombinant colonies are easily selected on the Agar plate.

In (X-gal) Lac Z Agar Plate

• Only transformed Colonies can grow in ampicillin medium, because it has plasmid with (AmpR) gene
• The beta -Z-galactosidase enzyme turns the medium on the plate blue
• Non-recombinant DNA means that these is no function and recombinant are white

Explore the process of cloning, which creates genetically identical copies of biological entities, including genes, cells, tissues, and organisms. Learn about the applications of cloned DNA in gene function analysis, mutation impact studies, and protein production. Discover the different types of artificial cloning: gene, reproductive, and therapeutic.