Lecture 27-Analytical Tools in Molecular Biology Flashcards
Restriction enzymes are named based on _____.
- what bacteria they’re expressed in
What two ways can restriction enzymes cut and what do each result in?
- asymmetrically: 5’ sticky ends
- symmetrically: blunt ends. Usually from cutting in palindromes
Restriction Modification System
- bacterial system that protects host from restriction enzymes via methylation of the recognition sequence by DNA methylase
Type I restriction enzyme (3)
- cleaves ~1000bp from recognition sequence
- endonuclease and methylase activities
- requires ATP
Type II restriction enzyme (3)
- cleaves very specific sequences by recognizing palindromes
- does NOT require ATP
- used in research because of their specificity
Type III restriction enzyme (3)
- cleaves ~25bp from recognition sequence
- endonuclease and methylase activities
- uses ATP
restriction enzymes allow us to make ______
recombinant DNA
pBR322
- first plasmid constructed
- has “unique” restriction sites (only one for each restriction enzyme (Pst1, EcoR1, BamH1, Sal1, PvuII
What kinds of ends does EcoR1 make?
- sticky
In order to insert a chromosomal segment into a plasmid you have to isolate both segments using _______
- the same restriction enzyme
What happens if there is not the restriction site you need on the plasmid?
- use a polysynthetic linker which contains many restriction sites and ligate it into the plasmid cloning vector with EcoR1 sites
Transformation (process)
NOTE: not 100% efficient because each step is not 100% efficient
- cleave cloning vector and chromosomal DNA with same restriction endonuclease
- ligate chromosomal DNA into vector to make the recombinant vector
- recombinant vector inserted into cell by keeping cell at 0º with CaCl2, then heat shocking it to open up the cell wall
- cell divides with varying amounts of plasmid (since it replicates independently of the host genome)
Why would you put a restriction site into an antibiotic resistance gene? Give an example using pBR322 and amp/tet resistance of why.
- a way to select for those bacteria that have the gene
- pBR322 is cleaved in the middle of the amp resistance gene by Pst1**
- foreign DNA is ligated into the plasmid disrupting the amp resistance, tet resistance remains intact because its in another spot on the plasmid
- cells transformed and grown on agar with tet for selection
- colonies that grow have plasmids so each colony is divided in half and put on either a tet only control agar plate or a tet + amp agar plate that will kill cells that DO have the gene of interest. Note that the colonies have to be placed in order to know which colony corresponds to which
- those that dont grow on the amp + tet plate but DO grow on the tet only plate have both the plasmid and the gene of interest
Why use bacteriophages instead of plasmids?
- contain more DNA (~60kb) so you can insert 20-40K bp vs. 200-2000 in plasmids
How do you use bacteriophage transformation?
- restriction nucleases cut out the center “filler” DNA
- foreign DNA fragments, also cut by the same restriction endonuclease mixed in and presumably the ends of the original bacteriophage genome attaches to new DNA. This is required because these parts are needed for viral packaging. If either of the ends are missing or if the filler segment is too small it won’t go into the bacteriophage
- DNA is packaged into lambda bacteriophages
What is the basic difference between a plasmid and a BAC plasmid? (2)
- BACs have par gene
- BACs have chloramphenicol resistance
- BACs contain multiple restriction sites (or cloning sites) for multiple restriction enzymes in lacZ gene