11.1 Restriction Endonuclease? More like restriction endopartylase!! Flashcards
what do type 1 restriction endonuclease do?
- they recognize specific DNA sequences and cleave DNA somewhere else
- restrict entry of foreign DNA into the cell
- originally thought to be rare, now known to be common (not very helpful in molecular biology)
what question was answered by T1 ER?
- why bacteriophages could grow onsite strains but not on others
what are type 2 restriction endonucleases?
- discovered in the 70s, thousands now available
- cleaves within the restriction site (on palindromic sequences)
- incredibly useful (can target where they want to cut)
why is type 2 more useful than type 1 in molecular biology?
type 1 is more unpredictable
what is a palindromic sequence?
- reads the same way in the 5’ to 3’ direction
In what order are the REs isolated?
- ECORI: 1st endonuclease isolated from the strain
- BamH: 1st endonuclease isolated from the strain
- HindIII: 3rd endonuclease isolated from the strain
why don’t restriction endonucleases destroy their own DNA?
- the host methylates a base in every copy of the RE site within its own genome
How are sites cut by RE II rejoined?
with ligase!
- many commercially available
what is gel electrophoresis?
- a method of separating DNA strands based on size
- at neutral pH DNA is negatively charged due to the phosphodiester bonds, so it travels towards the positive electrode
Can electrophoresis be performed in a liquid?
no way, no how
- needs to be a gel
what gel is used for electrophoresis?
- most common gel is agarose made from agar agar seaweed
- it’s an uncharged polysaccharide
- agar, water, and a buffer which keeps the pH slightly above neutral and allows for current flow
What are the steps for preparing an agar plate?
- barriers placed into gel tray
- molten agarose placed in tray
- insert combs to form solid wells before the agar hardens
- load DNA into wells and apply voltage
how is the size fractioned DNA in gel electrophoresis visualized?
- EtBr is a binding fluorescent dye : it’s intercalating so may cause frameshift
- stains used now binds to minor groove
what curve can be used to extrapolate size from electrophoresis?
- a standard curve can be used: larger molecules / more base pairs travel more slowly and a smaller distance
- often the first step in DNA identification
what factors impact the rate of mobility in DNA fragments in gel?
- size
- agarose concentration in gel
- topology (physical conformation)
- Voltage
how might concentration of agarose effect migration rate?
- as agarose concentration increases, pore size decreases
- more resistant to molecular movement, so benefits smaller molecules and the differences of one base pair can be detected
How do topologies impact molecular movement of fragments?
- linear, relaxed, supercoiled
- topology impacts rate of migration in cells
- relaxed goes slower, supercoiled goes further
What is supercoiled DNA? are cells positively or negatively supercoiled?
- circular or linear, but the linear ends must be restrained
- mostly negatively supercoiled, but positively supercoiled can be produced in vitro
How does voltage impact the migration of molecules?
- further migration in greater voltage
which factors do NOT impact the rate of migration of molecules in electrophoresis?
- the % of GC content and the concentration of DNA
- equal rates for both regardless
Why is Type II RE so useful?
- more predictable and can be modified to cut specific sequences
How did restriction endonucleases revolutionize molecular biology?
- 1st easy way to study variation and map particular features
- 2) for first time could easily recombine DNA sequences I(ligase) and create novel DNA sequences = recombinant DNA!
(also easier lab methods)
