GeneticsCh20

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).

• 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.

• 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

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

• 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.

Yeast most common for euk genes

• 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

• Humulin (insulin)
• Erythropoitin
• Human growth hormone
• Hepatitis B vaccine

• Herbicide-resistance corn/soybeans
• Golden rice
• virus-resistant papaya

• 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

• 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