Polymerases Flashcards
For the following: what is the template, synthesised strand & longer name?
- DNA polymerase
- RNA replicase
- RNA polymerase
- Reverse transcriptase
What do terminal deoxynucleotidyl transferase TdT do?
What kind of polymerase are they?
What do poly(A) polymerase do?
What kind of polymerase are they?
- DNA & DNA - DNA-dependent DNA polymerase
- RNA & RNA - RNA-dependent RNA polymerase
- DNA template, RNA strand - DNA-dependent RNA polymerase
- RNA template & DNA strand - RNA-dependent DNA polymerase
String non-specific bases onto 3’ end of DNA (DNA dependent DNA polymerase)
Add 3’ poly(A) tail onto mRNA after transcription - RNA-dependent RNA polymerase
What are the 3 activities of DNA polymerase I?
How was Klenow formed and why was it used in molecular biology?
Why is Klenow no longer used?
What was Klenow used for?
5’ to 3’ catalytic, 3’ to 5’ exonuclease proofread, 5’ to 3’ exonuclease (RNA primer removal- nick translation)
Digestion of Pol I with subtilisin protease. Use as lacks 5’ to 3’ exonuclease activity so doesn’t degrade RNA primers (made by primase on lagging strand)
Denatured at high temperatures (add fresh Klenow every cycle), couldn’t distinguish between dNTPs & ddNTPs, stalled at secondary structures, low processivity
Sanger sequencing, filled in 3’ ends to make blunt ends, digested 3’ ends away to make blunt ends, labelling with radioactive bases and making radioactive DNA probes
Why was Taq polymerase such an advance in Molecular Biology?
What was its temperature drawback?
Why does Taq have low fidelity?
What does this mean for molecular biology use?
More thermostable, higher processivity, faster synthesis due to lack of 3’ to 5’ exonuclease
Short half-life at 100C for deannealing temperatures
Lack of 3’ to 5’ proof reading means higher error rates & exhibits TdT activity by adding adenine onto end of 3’
Complicated in DNA cloning with extra A on 3’ end
Why is Pfu better than Taq?
What is its drawback?
What is Phusion polymerase?
What features does it have?
Higher thermotolerance, lower error rate (3’ to 5’ exonuclease)
Slower synthesis rate due to 3’ to 5’
Enzyme fused with DNA-binding/processivity-enhancing domain
High synthesis rate, high fidelity (accuracy), high thermostability
What can you use TdT for to join DNA?
What else could you program TdT for?
Produce homopolymer tails - introduce TdT with one type of dNTP to string on end
Sequence DNA
Do RNA polymerases need a primer?
What do SP6 & T7 RNA polymerase require?
What subunits make up E. coli RNA polymerase?
Why are T7 RNA polymerases more used?
No, just a DNA template. DNA polymerases need 3’OH
Mg2+, double stranded template & specific promoter
2 alpha, 2 beta, omega & sigma factor
1 subunit, cheaper, high specificity for promotor, high fidelity
What does reverse transcriptase require?
What 3 ways can you do this?
What can you use as a template?
What is the end product?
What considerations do you need when choosing a RT?
Primer - as forming DNA
gene specific priming, oligodT priming, random hexamer priming
RNA or ssDNA
cDNA -> amplified to make cDNA libraries
Need high fidelity (lack of errors), high thermostability, high processivity
