AMB2 Flashcards
(26 cards)
p(sub)GATC OH(sub)GATC
What does this nomenclature mean?
pGATC = phosphporylated at 5' end OHGATC = dephosphorylated
starting from phosphate attatched to 5’ end of sugar what are the names of the phosphates
alpha, beta, gamma
What are 5 features of type 2 restriction enzymes
- seperate endonuclease and methylase activity
- Mg2+ cofactor
- cut at defined positions
- most recognise symmetric DNA seqs and bind as homodimers
- few recognise assymetirc and bind as heterodimers.
name 3 type 11 restriction enzymes
EcoR1
Pst1
Hae111
What is EcoR1 restriction site and where does it cut
GAATTC
what is pst1 restriction site and where does it cut
CTGCAG
cleaves between AG
free phosphate at 5’ end
‘recessed’
what is Hae111 restriction site and where does it cut
GGCC
cleaves in centre GC - blunt ends
free phosphate at 5’ end
what is an isochizomer
enzyme that recognise the same sequence but originate from different organisms. may cut at different points.
what does bacteriophage T4 ligase do?
ligation of cohesive dna terminals or nicks, can act on dna and rna
what does t4 ligase need to function
mg2+ cofactor
ATP
and substrates need to be phosphorylated at the 5’ end.
- more efficient if substrates have sticky ends. but capable of joining together blunt ends.
What does T4 polynucleotide kinase do?
adds phosphate group to desphosphorylated molecule
- could radiolabel using radioactive phosphate group.
- more efficient in dsDNA than ss however with sufficient ATP can be phosphorylated
what does alkaline phosphatase do?
catalyses nucleotide dephosphorylation - opposite of kinase.
where is alkaline phosphatase extracted from?
ussually eukaryotic sources,
calf intestine
or shrimp - much less heat stable.
what is alkaline phosphatase used in?
cloning procedures eg. inhibition of ligation.
What activities does E.coli DNAP1 (holoenzyme) have?
5’ -> 3’ polymerase = substrate ssDNA and primer with 3’OH
5’ -> 3’ exonuclease = degrades RNA/DNA or RNA:DNA in absensce of nucleotides.
3’->5’ exonuclease = if has 3’OH will degrade from 34’ but this activity is blocked by 5’->3’ polymerase activity and inhibited by dNMP’s with 5’p
what happenes if DNAP1 is active in the presence of DNA and only one dntp
3’->5’ exonuclease activity will degrade dsDNA from 3’ until base exposed that is complementary to single nucleotide and a series of exchange and synthesis reactions occur at that poistion - used in ligation indep cloning.
What is the klenow fragment?
DNAP without 5’ exonuclease activity
3’ -> 5’ activity can be controlled by dNTPs
What activity does Reverse Transcriptase have?
5’->3’ DNAP with RNA or DNA as template primer baring 3’)H
RNAse H 5’->3’
3’->5’ exoribonuclease activity.
degrades RNA in RNA:DNA heterduplex from either 5’ or 3’ end.
What are SP6, T3 and T7 RNAPs?
5’->3’ DNA dep RNAP commonly used to synthesize RNA in vitro.
recognice simple short dsDNA seqeunces as promoters
Whar are nucleases?
enzymes that degrade nucleic acids
what are exonucleases, give two examples
degrade dsDNA from both/either sides
Bal31 = degrades from 3’ and 5’ end
exo111 = degrades from 3’ end leaving 5’ overhang
what are endonucleases give 2 examples
degrade dsDNA from inside polymer
S1 nuclease = degrade only ss DNA and dsDNA if there is a nick
DNAse1 - generic, degrades all DNA to comletion.
In electrophoresis what colours are the pos and neg sides?
pos - red
neg - black
in electrophoresis what does speed of migration depend on?
size of fragmnent
strength of field
conc of agarose
