Feb 25 Flashcards
(22 cards)
When nuclease cuts, which 5’ or 3’ phosphatase is more commonly used?
The 5’ is more commonly used
When ribonucleases cut, what diff from deoxyribonucleases?
Ribozymes, so have 2’ OH on sugar as well, they use it to attack phosphate, so get 2’,3’ cyclic monophosphate, instead of either 3’ or 5’ phosphate for deoxy.
What is template, bonds broken, products, where cut, and uses of micrococcal nuclease (MNase)?
It cuts everything, both ss and ds DNA and RNA. It is non specific, cuts single strandedly. It is both endo and exonuclease. Leaves 3’ phosphate. Used to determine areas where DNA is wrapped around histones.
What is template, bonds broken, products, location cut, and uses of Mung Bean nuclease?
It will cut only single stranded DNA and RNA. It is exonuclease both 3-5’ and 5’-3’. Leaves 5’ phosphate. Used to remove both 5’ and 3’ overhangs so create blunt ends.
What is template, bonds broken, products, location cut, and uses of DNase 1/2?
They nonspecifically cut ss or ds DNA. Make single stranded cuts. DNase 1 leaves 5’ phosphate, DNase 2 leaves 3’. Used to remove DNA so can isolate RNA. Also can determine areas of open chromatin
What do type 2 restriction endonucleases recognize?
They typically recognize specific sequences ( usually palindromic)
Do all restriction endonucleases leave overhangs?
No, some leave blunt ends.
When have restriction endonuclease cleavage site, how far from end of DNA should you aim for?
At least 6 base pairs as some will be less effective at the end of DNA
What is restriction endonuclease star activity? What can cause?
It is when RE’s lose specificity under non-standard conditions. High glycerol, diff buffer, long rxn time
How does restriction endonuclease site methylation affect cutting? What methyltransferases?
It is dam an dcm methyltransferases that recognize and methylate DNA sequences. It means RE can’t cut at that spot, so use bacterial strain with those genes knocked out.
What is template and bonds broken, products and uses in lab of RNase A?
It cuts ss and dsRNA, endonuclease that cuts after pyrimidine, single stranded cut. Leaves 2’,3’ cyclic monophosphate. Used to remove RNA from DNA sample
What is template and bonds broken, products and uses in lab of RNase H?
It will cut RNA when in a duplex with DNA, so removes okazaki framents. It is non specific, single stranded cut, leaving 5’ phosphate. So destroys RNA template after making cDNA
What ATP used for for ligase?
It is used for energy only
What does T4 polynucleotide kinase do?
It will phosphorylate ss and ds DNA and RNA on the 5’ OH, using gamma phosphate of ATP. Can be used for cloning and to radiolabel DNA.
What do alkaline phosphatases do?
Will remove both 5’ and 3’ ends of ss, ds, DNA and RNA. SO prevents ligation of linearized plasmid DNA
What is other word for RNA dependent DNA polymerase?
Reverse transcriptase
What is processivity and fidelity for polymerase?
Processivity is how many nucleotides processed in a single binding event. Fidelity is how accurate it is, if has 3’ to 5’ exonuclease activity, it can backspace if incorrect base pairing.
DNA Pol 1 vs Klenow Fragment?
Pol 1 is 5’-3’ polymerase, it has exonuclease activity in both directions, so can removve existing nucleotides in front and behind it. Klenow will lose the 5’ to 3’ exonuclase as it is a framgment of DNA Pol 1, so will fill missing bases but not replace existing ones
What is T4 DNA polymerase?
No 5’ to 3’ exonuclease activity like Klenow fragment, but has stronger 3’ to 5’ exonuclease activity adn higher processivity
Do RNA polymerases need a primer?
No uses DNA template but no primer needed, makes mRNA, or NRA etc
What does terminal deoxynuclotidyl transferase do?
It will add whatever nuclotides to 3’OH end of DNA moleulces. SO if given only A, it will make poly aA tail etc. Does not need primer. WIll do both ss and ds templates, blunt or overhangs
