What are restriction enzymes and how do they work?
Restriction enzymes are bacterial enzymes that recognize a specific sequence (restriction site) and cut both strands of DNA at that sequence. Most recognition sites are a palindrome (RCCR).
What is the difference between staggered end cut and blunt end cuts made by restriction enzymes?
- Staggered-end cuts: have single-stranded overhangs. These can base pair with complementary staggered-end cuts cut using the same restriction enzyme and anneal.
- Blunt-end cuts: have double stranded ends. These do not have an area for hybridization.
How is DNA fragmented and separated on gel following restriction enzyme treatment?
- Restriction mapping: Establishes the number of, order of, and distances between restriction-enzyme cleavage sites along a cloned segment of DNA.
- Created by cutting DNA with different restriction enzymes and separating by gel electrophoresis (smallest fragments moves farthest through gel) creating a unique series of bands.
- Southern blotting: 1) separation of DNA fragments by gel electrophoresis 2) hybridization using labeled probes.
- Northern blotting: mRNA blotting (actively expressed genes)
- Western blotting: analyzing proteins
What are RFLPs?
restriction fragment length polymorphisms: variations in the short base sequences where restriction enzymes can cut. Comparisons can be made between two samples cut with the same restriction enzyme
What are the differences between these vectors, and what hosts would they be introduces to? plasmid, YAC, BAC, HAC, Ti Plasmid, Cosmid
- Plasmid: introduced to bacteria via transformation. Plamsid and DNA are cut with same restriction enzyme, then allowed to anneal. Many copies per bacteria. Blue-white selection is used to determine which bacteria have the desired product. Too small for some larger genes.
- YAC: yeast artificial chromsomes. Can clone large-fragments of DNA. Linear, has telomers, origins or rep, centromere.
- BAC: bacterial articificial chromsomes. Large, but low copy (1-2 per cell) plasmids.
- Ti Plasmid: Plants. Plasmid recovered from Agrobacterium. Segment of DNA from plasmid is transfered to plant cell genome. Tumor-causing elements removed from T-DNA.
- Cosmid: Created from plasmid AND virus components.
Bacteria most common for prok genes
Yeast most common for euk genes
What is required of a DNA cloning vector?
- Several restriction sites that allow insertion of the DNA fragments to be clones
- intoduced into host cells to allow for independent replication of the DNA fragment
- Selectible marker gene to show they have been taken up
- Easy to isolate from host cell for DNA recovery
What are some examples of medicinal products produced by recombinant DNA technology?
- Humulin (insulin)
- Human growth hormone
- Hepatitis B vaccine
What are some examples of genetically modified crops?
- Herbicide-resistance corn/soybeans
- Golden rice
- virus-resistant papaya
What are some selection methods used to screen for host cells that have taken up the recombinant DNA molecules?
- Blue/white selection: The only bacteria that grow have taken up a plasmid (ampicillin resistance), blue colonies have a working lacZ gene and do not contain the desired DNA, white colonies have a disrupted lacZ gene and contain the desired product.
- Growth on antibiotic medium
Explain how biotechnology products can be produced by using recombinant DNA technology
- 1) the appropriate vector is determined based on size and type of gene
- 2) the gene and vector are cut using the same restriction enzyme and attempted to be hybridized
- 3) the hybridized vector is inserted into its appropriate host
- 4) the host accepts the vector and vector replicates the gene product
- 5) a selection is made using the selective marker to determine which hosts have the foreign DNA
- 6) these hosts are harvested to retrieve the desired product