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Microbiology
Chapter 11 Manipulation of DNA
| Term | Definition |
|---|---|
| Genetic Engineering | The use of in vitro techniques to alter genetic material in the laboratory |
| Restriction Enzymes | Recognize specific DNA sequences and cuts DNA at those sites Widespread among prokaryotes Protect prokaryotes from hostile DNA(viral genome) |
| Type 2 Restriction Enzyme | Cleave DNA Have sticky or blunt ends |
| Modification Enzymes | Protect cells DNA from restrictio enzymes by chemically modifying nucleotides in restriction recognition sequence Usually consists of methylation of DNA |
| Gel Electrophoresis | Separated DNA based on size Nucleic acids migrate towards + end due to - PO3 groups |
| Nucleic Acid Hybridization | Base pairing of a single strand of DNA or RNA to give hybrid double helix |
| Nucleic Acid Probe | Segment of single stranded DNA that is used in hybridization and has predetermined identity |
| Southern & Northern Blot | A hybridization procedure where DNA is in the gel and probe is DNA or RNA Northern: RNA is in the gel |
| Molecular Cloning | Isolation and incorporation of a piece of DNA into a ector so it can be replicated and manipulated |
| 3 Steps of Molecular Cloning #1 | Isolation and fragmentation of source DNA Source can be genomic DNA RNA or PCR amplified fragments Genomic DNA must first be digested |
| 3 Steps of Molecular Cloning #2 | Insertion of DNA fragment into cloning vector Most vectors derived from plasmid or virus |
| 3 Steps of Molecular Cloning #3 | Introduction of cloned DNA into host organism Transformation: some cells will contain desired gene |
| Gene Library & Shotgun cloning | Mixture of cells containing a variety of genes Gene libraries made by cloning random genome fragments |
| Essentials of Molecular Cloning | Essential to detect right gene Initial screen, may need to look closer if working with heterogenous gene library |
| Antibodies | Blood serum proteins produced by animals Made by injecting animal with specific protein antigen |
| Reporter Genes | Encodes proteins that are easy to detect and assay (GPC, LacZ) |
| Gene Fusions | Promoters or coding sequences of genes of interest can be swapped with those of reporter genes to elucidate gene regulation under various conditions |
| Plasmids | Plasmids are natural vectors useful as cloning vectors Small size; easy to isolate DNA Independent origin of Replication Multiple copy number Presence of selectable markers Vector transfer carried out by chemical transformation or electroporation |
| PUC19 | Common cloning vector Modified E. coli plasmid |
| What genes does PUC19 carry? | Ampicillin resistance and lacZ genes |
| When is lacZ inactivated? Why? | Inactivated by insertion of foreign DNA Cant process B-galactadase and blue color does not show in screens |
| The ideal host for cloning vectors should be? | Capable of rapid growth in inexpensive medium Capable of incorporating DNA Genetically stable in culture Equipped with appropriate enzymes to allow replicatio of vector |
| Shuttle Vectors | Vectors that are stably maintained in 2 or more unrelated host organisms |
| How do you engineer a plasmid to function in eukaryotes? | Add a eukaryotic origin of replication add a centromere recognition sequence |
| Expression Vectors | Allow experimentor to control the expression of cloned genes Based on transcriptional control Allows for high levels of protein expression Strong promoters Effective tralnscription terminators used |
| What does the T7 promoter expession vector do? | Cloned genes are under control of T7 promoter Gene for T7 RNA polymerase recognizes only T7 promoters Transcribes only cloned genes Shuts down host transcription |
| Why are there problems with translating DNA into eukaryotes? | Bacterial ribosome binding sites are not present Differences in codon useage between organisms Eukaryotic genes containing introns will not be expressed properly in prokaryotes |
| Integrating vectors | Integrate into host chromosome Stably maintained in cell |
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