Genetic Engineering and Gene Cloning Lecture Notes PDF

# Genetic Engineering ## Human Genome - 3.2 billion bases ## Biotechnology Today - Genetic Engineering - Manipulation of DNA - If you are going to engineer DNA & genes & organisms, then you need a set of tools to work with. - This unit is a survey of those tools. ## Bacteria - Bacter...

# Genetic Engineering ## Human Genome - 3.2 billion bases ## Biotechnology Today - Genetic Engineering - Manipulation of DNA - If you are going to engineer DNA & genes & organisms, then you need a set of tools to work with. - This unit is a survey of those tools. ## Bacteria - Bacteria review - One-celled prokaryotes - Reproduce by: - Binary fission - Rapid growth - Generation Every ~20 minutes - 10^8 (100 million) colony overnight! - Dominant form of life on Earth - Incredibly diverse ## Bacterial Genome - Single circular chromosome - Haploid - Naked DNA - No histone proteins - ~4 million base pairs - ~4300 genes - 1/1000 DNA in eukaryote ## Transformation - Bacteria are opportunists - Pick up naked foreign DNA wherever it may be - Have surface transport proteins that are specialized for the update of naked DNA - Import bits of chromosomes from other bacteria - Incorporate the DNA bits into their own chromosome ## Plasmids - Small extrachromosomal circular DNA - 5000 - 20,000 base pairs - Self-replicating - Carry extra genes - 2-30 genes - Genes for antibiotic resistance - Can be exchanged between bacteria ## How can plasmids help us? - A way to get genes into bacteria easily - Insert new gene into plasmid - Insert plasmid into Bacteria - vector - Bacteria now expresses new gene - Bacteria make new protein ## Biotechnology - Plasmids used to insert new genes into bacteria ## Engineered Plasmids - Building custom plasmids - Restriction Enzyme sites - Antibiotic resistance genes as a selectable marker ## Selectable Marker - Antibiotic resistance gene on plasmid - Ampicillin resistance - Selecting for successful transformation - Successful uptake of recombinant plasmid ## Selection for plasmid uptake - Antibiotic becomes a selecting agent - Only bacteria with the plasmid will grow on antibiotic (ampicillin) plate ## Screening for Recombinant Plasmid - Bacteria take up plasmid - Functional LacZ gene - Bacteria make blue color - Bacteria take up recombinant plasmid - Non-functional LacZ gene - Bacteria stay white color ## How Do We Cut DNA? - Restriction enzymes - Restriction endonucleases - Discovered in 1960s - Evolved in bacteria to cut up foreign DNA - Bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA ## Restriction Enzymes - Action of enzyme - Cut DNA at specific sequences - Restriction site - Symmetrical - Produces protruding ends - Sticky ends - Will bind to any complementary DNA - Many different enzymes - Named after organism they are found in - EcoRI, HindIII, BamHI, Smal ## Restriction Enzymes - Cut DNA at specific sites - Leave "sticky ends" ## Sticky Ends - Cut other DNA with same enzymes - Leave "sticky ends" on both - Can glue DNA together at "sticky ends" ## Sticky Ends Help Glue Genes Together - DNA ligase joins the strands. - Sticky ends stick together ## Why Mix Genes Together? - Gene produces protein in different organism or different individual ## Copy (& Read) DNA - Transformation - Insert recombinant plasmid into bacteria - Grow recombinant bacteria in agar cultures - Bacteria make lots of copies of plasmid - "cloning" the plasmid - Production of many copies of inserted gene - Production of "new" protein - Transformed Phenotype - DNA → RNA → protein → trait ## Grow Bacteria...Make More ## Cut, Paste, Copy - Word processing metaphor... - Cut - Restriction enzymes - Paste - Ligase - Copy - Plasmids - Bacterial transformation ## Gene Cloning ## Gene Cloning - What does the term cloning mean? - What is gene cloning? How does it differ from cloning an entire organism? - Why is gene cloning done? - How is gene cloning accomplished? ## Cloning Definition - A method of growing an individual from a single somatic cell of its parent - Clone: a collection of molecules or cells, all genetically identical to an original molecule or cell. ## What is DNA/gene cloning? - When DNA is extracted from an organism, all its genes are obtained - In gene (DNA) cloning a particular gene is copied (cloned) ## What is Gene cloning? - To "clone a gene" is to make many identical copies of it. - Gene can be an exact copy of a natural gene. - Gene can be an altered version of a natural gene. ## Why Clone DNA? - A particular gene can be isolated and its nucleotide sequence determined - Sequences of DNA can be identified & analyzed - Protein/enzyme/RNA function can be investigated - Mutations can be identified, e.g. gene defects related to specific diseases - Organisms can be 'engineered' for specific purposes, e.g. insulin production, insect resistance, etc. ## How is DNA cloned? - Cell-based DNA cloning - Cell-free DNA cloning (PCR) ## Cell Based DNA Cloning/ Clone in Dividing Cells - Double-stranded recombinant plasmid DNA introduced into bacterial cell ## Restriction Endonucleases - The Molecular Scissors - Host enzymes that prevent the invasion of foreign DNAs such as viral DNA, by cutting them up. - These enzymes cut within the foreign DNAs, rather than chewing them away from the ends. - These enzymes recognize a specific DNA sequence (4-12bp) and cut both DNA strands. ## Three Types of Restriction Enzymes - Type I, Type II, Type III - Type I and III - Cleave with less precision and are not used in manipulating DNA - Type II - Recognize specific DNA sequences - Cleave at specific site within sequence - Can lead to "sticky ends" that can be joined. ## DNA ligase covalently links two DNA strands ## DNA ligase - Joins the two fragments forming a stable DNA molecule - Catalyzes formation of a phosphodiester bond between adjacent phosphate and hydroxyl groups of DNA nucleotides. ## Cloning Vectors - The DNA Carriers - Plasmids (e.g. pBR 322, pUC 19) - Phages/Bacteriophages (viruses that infect bacterial cells e.g. lambda phage) - Artificial chromosome vectors, e.g. - YACs (yeast artificial chromosomes) - BACs (bacterial artificial chromosomes) - Phagemids (combined features of phages with plasmids e.g. pBluescript II KS) - Cosmids (plasmid vectors with COS site of phages) - Viral vectors (retiroviruses) - Retriever vectors - Shuttle vectors ## Plasmids that can be modified to carry new genes - Plasmids useful as cloning vectors must have: - Replicator (origin of replication) - Allow the vector as well as the foreign DNA to amplify in the host cell - Selectable marker (antibiotic resistance gene) - Allow the host to grow on selective media - Can selectively amplify this specific vector in the host cell - Multiple cloning site - Allow insertion of foreign DNA without disrupting replication or inactivating essential markers ## DNA can be inserted into a cell by: - Transformation - Electroporation - Protoplast fusion - Microinjection - Biolistics/Gene gun ## The first Cloning experiment done by Boyer and Cohen ## Inserting a DNA sample into a Plasmid ## Screening ## Propagation - Once colonies are identified, they are cultured in broth to increase numbers and therefore the amount of DNA. - Samples are also prepared for storage at -80 degrees. They can be kept for many years this way.