Biotech Q3 Exam Review PDF

# Genetic Engineering Genetic engineering is defined as the direct manipulation of an organism's genes including heritable and non-heritable recombinant DNA constructs. ## GENES VS. DNA - Genes are segments of the DNA which give you physical characteristics that make you unique; carry instruction...

# Genetic Engineering Genetic engineering is defined as the direct manipulation of an organism's genes including heritable and non-heritable recombinant DNA constructs. ## GENES VS. DNA - Genes are segments of the DNA which give you physical characteristics that make you unique; carry instructions that tell your cells how to work and grow. - DNA, a nucleic acid which is responsible for building and maintaining the human structure. ## What is a TOOL or TECHNIQUE? A device or instrument used to carry out a particular function. ## What are some products of Genetic Engineering? - GMO (Genetically Modified Organisms) Crops - apple mango, seedless grapes, canola oil, corn oil # 11.1 Restriction and Modification Enzymes - Genetic engineering: using *in vitro* techniques to alter genetic material in the laboratory. - Basic techniques include: - Restriction enzymes - Gel electrophoresis - Nucleic acid hybridization - Nucleic acid probes - Molecular cloning - Cloning vectors - Restriction enzymes: recognize specific DNA sequences and cut DNA at those sites. - Widespread among prokaryotes - Rare in eukaryotes - Protect prokaryotes from hostile foreign DNA (e.g., viral genomes) - Essential for *in vitro* DNA manipulation - Three classes of restriction enzymes - Type II cleave DNA within their recognition sequence and are most useful for specific DNA manipulation. - Restriction enzymes recognize inverted repeat sequences (palindromes) - Typically 4-8 base pairs long; EcoRI recognizes a 6-base-pair sequence. - Sticky ends or blunt ends - Restriction enzymes protect cell from invasion from foreign DNA - Destroy foreign DNA - Must protect their own DNA from inadvertent destruction - Modification enzymes: protect cell's DNA for restriction enzymes - Chemically modify nucleotides in restriction recognition sequence - Modification generally consists of methylation of DNA. - Gel electrophoresis: separates DNA molecules based on size. - Electrophoresis uses an electrical field to separate charged molecules - Gels are usually made of agarose, a polysaccharide - Nucleic acids migrate through gel toward the positive electrode due to their negatively charged phosphate groups - Gels can be stained with ethidium bromide and DNA can be visualized under UV light - The same DNA that has been cut with different restriction enzymes will have different banding patterns on an agarose gel - Size of fragments can be determined by comparison to a standard - Restriction map: a map of the location of restriction enzyme cuts on a segment of DNA # 11.2 Nucleic Acid Hybridization - Nucleic acid hybridization: base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix. - Segment of single-stranded DNA that is used in hybridization and has a predetermined identity is called a nucleic acid probe - Southern blot: a hybridization procedure where DNA is in the gel and probe is RNA or DNA. - Northern blot. RNA is in the gel. # 11.3 Essentials of Molecular Cloning - Molecular cloning: isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated. - Three main steps of gene cloning: 1. Isolation and fragmentation of source DNA 2. Insertion of DNA fragment into cloning vector 3. Introduction of cloned DNA into host organism - Isolation and fragmentation of source DNA - Source DNA can be genomic DNA, RNA, or PCR-amplified fragments. - Genomic DNA must first be restriction digested - Insertion of DNA fragment into cloning vector - Most vectors are derived from plasmids or viruses - DNA is generally inserted *in vitro* - DNA ligase: enzyme that joins two DNA molecules - Works with sticky or blunt ends. - Introduction of cloned DNA into host organism - Transformation is often used to get recombinant DNA into host - Some cells will contain desired cloned gene, while other cells will have other cloned genes. - Gene library: mixture of cells containing a variety of genes. - Shotgun cloning: gene libraries made by cloning random genome fragments. - Essential to detect the correct clone - Initial screen: antibiotic resistance, plaque formation - Often sufficient for cloning of PCR-generated DNA sequences - If working with a heterogeneous gene library you may need to look more closely. # 11.4 Molecular Methods for Mutagenesis - Synthetic DNA - Systems are available for *de novo* synthesis of DNA - Oligonucleotides of 100 bases can be made - Multiple oligonucleotides can be ligated together - Synthesized DNA is used for primers and probes, and in site-directed mutagenesis - Conventional mutagens produce mutations at random - Site-directed mutagenesis: performed *in vitro* and introduces mutations at a precise location. - Can be used to assess the activity of specific amino acids in a protein - Structural biologists have gained significant insight using this tool - Cassette mutagenesis and knockout mutations - DNA fragment can be cut, excised, and replaced by a synthetic DNA fragment - The process is known as cassette mutagenesis. - Gene disruption is when cassettes are inserted into the middle of the gene. - Gene disruption causes knockout mutations. # 11.6 Plasmids as Cloning Vectors - Plasmids are natural vectors and have useful properties as cloning vectors - Small size; easy to isolate DNA - Independent origin of replication - Multiple copy number; get multiple copies of cloned gene per cell - Presence of selectable markers - Vector transfer carried out by chemical transformation or electroporation - Blue/white screening - Blue colonies do not have vector with foreign DNA inserted - White colonies have foreign DNA inserted - Insertional inactivation: lacZ gene is inactivated by insertion of foreign DNA. - Inactivated lacZ cannot process Xgal; blue color does not develop # 11.7 Hosts for Cloning Vectors - Ideal hosts should be - Capable of rapid growth in inexpensive medium - Nonpathogenic - Capable of incorporating DNA - Genetically stable in culture - Equipped with appropriate enzymes to allow replication of the vector - Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae. # 11.8 Shuttle Vectors and Expression Vectors - mRNA produced must be efficiently translated and there are problems with this always happening - Bacterial ribosome binding sites are not present in eukaryotic genomes - Differences in codon usage between organisms - Eukaryotic genes containing introns will not be expressed properly in prokaryotes - Expression vectors: allow experimenter to control the expression of cloned genes. - Based on transcriptional control - Allow for high levels of protein expression - Strong promoters: lac, trp, tac, trc, lambda PL - Effective transcription terminators are used to prevent expression of other genes on the plasmid.