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Microbiology
Chapter 11 Manipulation of DNA
| Term | Definition |
|---|---|
| Genetic Engineering | The use of in vitro techniques to alter genetic in the laboratory |
| Restriction Enzymes | Recognize specific DNA sequences and cut DNA at those sites |
| Characteristics of Restriction Enzymes | Widespread in prokaryotes Protect prokaryotes from hostile DNA (viral genomes) 3 Classes |
| Type 2 Restriction Enzyme | Recognizes inverted repeat sequences palindromes Sticky or blunt ends 4-8bp long |
| Modification Enzymes | Protect cells DNA from restriction enzymes by chemically modifying nucleotides in restriction recognition sequence. Modification usually methylation |
| Gel Electrophoresis | Separates DNA based on size Electric field separates charged molecules Nucleic acids migrate towards + electrode because of PO3 groups |
| Nucleic Acid Hybridization | Base pairing of single strands of DNA or RNA to get hybrid double helix |
| Southern and Northern blot | DNA or RNA in a gel Probe is DNA or RNA |
| Molecular Cloning | Isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated |
| Molecular Cloning Step #1 | Isolation and Fragmentation of source DNA Source DNA can be genomic DNA, RNA or PCR amplified fragments. Genomic DNA must first be digested |
| Molecular Cloning Step #2 | Instertio of DNA fragment into a cloning vector Most vectors derived from a plasmid or virus |
| Molecular Cloning Step #3 | Introduction of cloned DNA into host organism Transformation is used to get recombinant DNA into host |
| 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 t detect correct clone;mInitial scren; may need to look closer if working with heterogenous library |
| Antibodies | Blood serum proteins produced by animals Made by injecting animal with specific protein antigen |
| Nucleic Acid Probe | Look for binding of labeled nucleic acid probe to DNA from specific colonies |
| Reporter Genes | Encodes proteins that are easy to detect and assay (GPC LacZ) |
| Gene Fusions | Promoters or coding sequuences of genes of interest can be swapped with those of reporter genes to elucidate gene regulation under various conditions |
| Plasmids | Natural vectors useful as cloning vectors |
| Why are plasmids useful as cloning vectors? | Small size easy to isolate DNA; Indpendent origin of replication;Multiple copy number;presence of selectable markers;vector transfer carried out by transformation or electroporation |
| PUC19 | Modified E. coli plasmid |
| What genes does PUC19 carry? | Ampicillin resistance and LacZ genes |
| Insertional Inactivation | LacZ gene is inactivated by insertin of foreign DNA Inactivation of LacZ by B-galactadase |
| Hosts for Cloning Vectors | Ideal host 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 vector |
| Shuttle Vectors | Vectors that are stably maintained in 2 or more unrelated host organisms |
| How do you engineer a bacterial plasmid to function in eukaryotes? | Add a origin of replication; Add a centromere recognition sequence |
| Expression Vector | Allow experimenter to control the expression of cloned genes Based on transcriptional control;Allow for high levels of protein expression;Stong promoter;effective transcription terminators used to prevent expression of other genes on plasmid |
| What Does T7 do? | Cloned genes are placed udner control of T7 promoter |
| How does the T7 promoter work? | Gene for T7 RNA polymerase is present and reognizes only T7 promoters; Transcribes only cloned genes;Shuts down host transcription |
| What are some of the problems with translating mRNA? | Bacterial ribosome binding sites are not present in eukaryotic genomes; Differences in codon useage between organisms; Eukaryotic genes containing introns not expressed properly in prokaryotes |
| Integrating Vectors | Integrate into host chromosome; Stably maintained in cell |
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