Lecture 5: restriction enzymes Flashcards
what do restriction enzymes do
cut double stranded DNA at specific sequences
how does a bacterium protect its own DNA
Some restrictiion enzymes cleave only at DNA recognition sites that are not methylated
how are restriction enzymes named
- They are named based on their origin
- They have the first letter of the bacteria’s genus, two letters of the species, the strain (if applicable), and the order of discovery
based on the rules of naming, what would this be Escherichia coli RY13, 5th identified
EcoRV
based on the rules of naming, what would this be Haemophilus aegypticus, 2nd identified
HaeII
how big are the cut sites made by restriction enzymes
They are usually 4 to 8 bases long, however they can be longer
true/false RE cut sites are usually palindromic
true
what are the types of ends that RE cut sites can produce
- blunt
- sticky
what are variable bases
- represent potential bases at one spot
- P= purine
- Y= pyrimidine
- W= weak
- S= strong
Which of the following DNA sequences are potential RE recognition sites?
a) 5’ GCACCACG 3’
b) 5’ GCACAGTGC 3’
c) 5’ GCACGTGC 3’
d) 5’ GCAACG 3’
c) 5’ GCACGTGC 3’
what is an isoschizomer
RE that cuts in a similar manner to another RE. They recognize the same recognition site
what is a neoschizome
RE that recognize the same cut but at different positions
what is the probabillity of occurrence of an RE
(1/4)^n
true/false Buffers for RE activity are provided by the company you order them from
true
what is the buffer composition for RE activity
pH and ionic strength requirements vary
what is the incubation temp for RE activity
Most work best at 37 degrees celsius but can be at higher temperatures.
what is heat inactivation in RE
the way to stop RE activity
true/false Restriction enzymes always only cleave their canonical sequences
- false
- under certain conditions, some restriction enzymes can cleave non-canonical sequences which are similar but different from usual recognition sequence
what triggers star activity
- Too much enzyme
- Wrong buffer
- Long reaction time
- High glycerol
- Organic solvents
true/false when using RE, the sticky ends need to be compatible to properly reform the DNA
true
joining 2 fragments cut by diff restriction nucleases is how much less efficient
10-100 times less
what is star activity
when RE cleave similar, but not identical, sequences to their canonical ones under non-standard conditions
what does linear restriction mapping do
characterize DNA by location of restriction enzyme sites
descrbe the steps in linear restriction mapping
- add up the sizes of the DNA fragments within each digestion
- analyze the single RE digestion w the smallest # of fragments first
- see the two potential cut spots … they will be identical
- overlay the other enzyme on top, flipping once again (we know its 3 and 7, but which side is which)
- then count how the pieces would divide, and see which map matches the predicted outcome
- idk look at slide 14+15 of her slides if you’re confused
describe the steps in circular restriction mapping
- add up fragments within each digest
- analyze the single digest w the largest number of DNA fragments always place one RE site at 12 o clock position
- place the location of the next RE so that the resultation DNA fragments will match those obtained by double RE digest
- idk look at slide 16 of her slides if you’re confused
