Molecular Cloning Lecture Notes PDF

Fei Geng NUCLEIC ACIDS Electrophoresis through a Gel Separates DNA and RNA Molecules According to Size   Gel electrophoresis separates DNA molecules according to their size (including molecular weight, shape, charge, topological properties etc.) subjected to an electric field through a gel m...

Fei Geng NUCLEIC ACIDS Electrophoresis through a Gel Separates DNA and RNA Molecules According to Size   Gel electrophoresis separates DNA molecules according to their size (including molecular weight, shape, charge, topological properties etc.) subjected to an electric field through a gel matrix reveal the bands by staining the gel with fluorescent dyes, such as ethidium Gel matrix Polyacrylamide  High resolving power  Separate DNA only over a narrow size range  Agarose  Less resolving power  Separate from one another DNA molecules of up to tens, and even hundreds, of kilobases  Polyacrylamide Agarose gel electrophoresis  DNA molecules (eg. entire bacterial or fungi chromosomes) can be resolved from one another with the electric field applied Plasmid Vectors Plasmids are circular pieces of DNA found naturally in bacteria.  Plasmids can carry antibiotic resistance genes, genes for receptors, toxins or other proteins.  Plasmids replicate separately from the genome of the organism.  Plasmids can be engineered to be useful cloning vectors.  Plasmid Vectors (continued)  Plasmid vectors can be designed with a variety of features:  Antibiotic resistance  Colorimetric “markers”  Multiple Cloning Region Antibiotic Resistance Markers Antibiotic Resistance Gene Multiple Cloning Region Multiple Cloning Region The cloning marker for this plasmid is the lacZ gene. Application of Plasmid Vectors In Molecular Cloning How it works? (a) Initially, the gene to be replicated is inserted in a plasmid or vector. (b) The plasmids are next inserted into bacteria by a process called transformation. (c) Bacteria are then grown on specific antibiotic(s). (d) As a result, only the bacteria with antibiotic resistance can survive and will be replicated. Plasmid Isolation from Bacteria How to rapidly isolate plasmid? (a) Inoculation and harvesting the bacteria (b) lysis of the bacteria (heat, detergents (SDS or Triton-114), alkaline(NaOH)), (c) neutralization of cell lysate and separation of cell debris (by centrifugation), Or other cell types Plasmid Extraction DNA Electrophoresis The process using electro-field to separate macromolecules in a gel matrix is called electrophoresis. DNA, RNA and proteins carry negative charges, and migrate into gel matrix under electro-fields. The rate of migration for DNA linear fragments is directly proportional to the voltage applied at low voltages. The migration rate of linear DNA fragments is a function of their length. In all cases, molecular weight markers are very useful to monitor the DNA migration during electrophoresis. Conformations of Plasmid DNAs Plasmid DNA may appear in the following three conformations: 1) "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut. Super Coiled SC 2) "Relaxed Circular" DNA is fully intact, but "relaxed" (supercoils removed). Relaxed region 3) "Linearized" DNA has both strands cut at only one site. Linear DNA Conformation of Plasmid DNAs The relative electrophoretic mobility (speed) of these DNA conformations in a gel is as follows: Linear (slowest) Relaxed Circular Supercoiled (fastest) Restriction Enzymes Restriction Enzymes (also called Restriction Endonucleases) are proteins that cleave DNA molecules at specific sites, producing discrete fragments of DNA. Restriction Enzymes (RE) were first isolated by Nathans and Smith in 1970. Why Restriction Enzymes?  Why would bacterial cells contain proteins that cleave DNA at specific sequences?  Generally restriction enzymes are thought to protect bacterial cells from phage (bacterial virus) infection. Bacterial cells that contain restriction enzymes can “cut up” invasive viral DNA without damaging their own DNA. 2.Restriction Endonucleases Cleave DNA Molecules at Particular Sites  Restriction Endonucleases ← Blunt end ↖ ← Sticky ends ↙ Digestion of a DNA fragment with endonucleases EcoRI Cloning a Piece of DNA " Cut plasmid vector with AvaI AvaI AvaI 5´ 3´ AvaI Excise DNA insert of interest from source using Ava I Ligate the insert of interest into the cut plasmid Ligation Ligation is the process of joining two pieces of DNA from different sources together through the formation of a covalent bond.  DNA ligase is the enzyme used to catalyze this reaction.  DNA ligation requires ATP.  Transforming Bacteria After you create your new plasmid construct that contains your insert of interest , you will need to insert it into a bacterial host cell so that it can be replicated.  The process of introducing the foreign DNA into the bacterial cell is called transformation.  Expressing your cloned gene  Expression of a cloned gene can be accomplished by:  The E. coli host  Mammalian cells (if the plasmid used is designed for expression in mammalian cells)  Using an in vitro using a cell-free system. (See education resources Unit 001: The relationship between genes and proteins) Application of Plasmid Vectors In Pharmaceutical and Agriculture Bioengineering One of the major uses of plasmids is to make large amounts of proteins. In this case, bacteria or other types of host cells can be induced to produce large amounts of proteins from the plasmid with inserted gene, just as the bacteria produces proteins to confer antibiotic resistance. This is a cheap and easy way of mass-producing a gene or the protein — for example, insulin, antibiotics, antobodies and vaccines. Green Algae for antibody production Transgenic Arabidopsis expressing GFP to study PDI functions Future Maize Crop Molecular farming for potential medical use Two-pronged corn kernels could provide a double dose of protein D. Gallie/UC Riverside 2004 Inbred B73 & Teosinte Vitamin C enhanced Corn, Gallie/UC Riverside 2003