Lecture 2 Flashcards
What are PCR rxns catalyzed by?
Polymerases
Generally use dna as template
All reactants needed for pcr
dNTPs
Polymerase
Primers
DNA template
Buffer w Mg2+
Why is Mg2+ needed for pcr
Cofactor for polymerase
DNA stability
Pcr cycle
Melt 98C 30s
Melt 98C 10s
Anneal 50C 30s
Extend 72C 90s
Extend 72C 10 min
What end does polymerase extend from?
3 prime oh that is free
Caveats of our study
-15 residues in binding protein
300 possible options for mutations
-higher order interactions ignored
-ignoring long distance effects
How many sets of primers needed for GA?
2
2 ga rxns simultaneous per side of DNA
Each primer has overhang on which of its ends?
5’
It’s 3’ end must be annealed and free for polymerase
For protein expression, where in vector to put insert?
Promoter
RBS = ribosome binding site
ATG start
What else is important for insert location?
Reading frame
When to remove stop codon from insert?
?
Is HCAII an example of convergent evolution?
YES
How many isoforms of human HCAII?
7
Isoform definition
?
What type of enzyme is HCAII
Metalloenzyme
What does zinc act as within enzyme?
Lewis acid
When zn2+ is coordinated by 3 neutral histidines, what does zn have a high affinity for?
Hydroxyl group
Acts as a nucleophile and attacks co2 -> carbonic acid
Steps for HCAII
?
RLS for HCAII?
Step 6
Base coming in for protein
Catalytic components
Glu 106
Thr 199
His 64
Stability components
His 94, 96, 119
Mutagenesis definition
?
Conservative vs no conservative definitions
?
Plasmid definition
?
Components of cloning
Ori = origin of replication = how vector clones itself
Selection marker = commonly an antibiotic resistant gene
MCS
- restriction enzyme site = site of snip = where insert can go ?
- promoter (T7)
- tags = many functions
Gibson Assembly
Cloning method that links and amplifies both vector and insertb
Roles of exonuclease, polymerase, and ligase in GA
?
Forward primer
Matches with written out gene exactly
Complement to anti sense strand
Reverse primer
Reverse complement to 3’ end of written out gene
Complement to sense strand
Sense strand
What is written out = gene standard
mRNA sequence
Template for translation
Anti Sense Strand
Actually encodes genetic info
Cloning definition
Clone gene into vector
Bp Range for primers
15-25bp
Free 3’ oh
RNA Virus and qPCR
?
Generic way a gene is written out
5’ to 3’
Sense strand
ATG
