4 - Molecular Biology Flashcards
molecular biology
techniques used to map DNA
techniques
- cutting
- pasting
- copying
restriction endonuclease
- cuts middle of DNA
- homodimers, therefore they can only cut palindromes
- evolve to cut a specific part of DNA, but it won’t be able to cut the newly formed DNA
sticky ends
- DNA dissociating after being cut
- can come together again if cooled
blunt ends
no overhang on the DNA cut
process of ligation
- NAD+/ATP cofactor is used to make a good LG in the E+
- the Nu- is able to attack the E+ with the DNA ligase
- sticky ends form non-covalent complex, then ligase will remake DNA and stick it back together
blunt end ligation vs. sticky end ligation
blunt end is much less efficient
plasmids
can be copied and amplified many times
plasmid contents
- circular DNA
- antibiotic
- ORI
- restriction sites
restriction sites
plasmid will contain a lot to give options for experimenters
plasmid replication
- insert DNA fragment into plasmid vector using restriction sites
- mix E. coli with plasmid (heat pulse/electrical shock)
- plate them in Petri dishes with ampicillin
- transformed cells will survive
- replicate plasmids that survived
- replicate cells that survived (plasmid + E. coli)
purification of plasmid DNA
- DNA sticks to silica really well
- mixture of silica with plasmid vector and DNA will separate the two into plasmid and DNA + silica
- adding water to DNA + silica mixture will separate the two and give pure DNA
polymerase chain reaction (PCR)
- most powerful techniques used
- uses 2 primers, therefore the amplification is exponential
- can be used to add restriction sites to the ends of a gene
ligating PCR products
- complementary restriction sites
- blunt edges
- TA overhangs
Gibson Assembly Cloning
- inserting DNA into plasmid without restriction sites
- requires T5 exonuclease
- can combine many fragments at once
- all happens in one step
- T5 exonuclease, DNA polymerase, and ligase all in one solution
T5 exonuclease
digests 5’ of DNA, leaving 3’ overhangs
Gibson Assembly Cloning process
- add overlap to one fragment with PCR
- use T5 exonuclease to digest 5’ ends, forming sticky ends
- recombine with non-covalent bonds
- use PCR to close gaps in fragments
- use ligase to close nicks
insert restriction site in DNA
- do two separate PCRs with the restriction site
- primer will have the restriction site - digest and ligate
problems with inserting restriction site with PCR
they can ligate to the other DNA strand through non-covalent bonds
site-directed mutagenesis
- do two separate PCRs with the mutation
- primer will have the mutation
- mutation will lower temperature as its base cannot pair with other strand - cool/lower the temperature
- add DNA polymerase for strands that cannot extend 5’ to 3’
- add outside primers and perform PCR to amplify desired mutation
random mutagenesis
doing a PCR reaction with manganese ion (instead of magnesium ion) and low concentration of one or more dNTPs will make errors within DNA
quickchange
- linear amplification, therefore NOT PCR
- most popular method of site-directed mutagenesis
quickchange process
- denature plasmid
- add primers with desired mutation
- add DNA polymerase until it hits nicks
- treat with Dpn1
Dpn1
endonuclease that will only digest the template DNA because only the template DNA is methylated
methylation of DNA from E. coli
methylate the amine of adenine
dam methylase
adenine N6 methyltransferase
