Exam 2 Review Flashcards
replication at the begining of mitosis results in what
sister chromatids, joined at centromere
what connects nucleosomes
histone h1
describe reaction of incoming nucleoside triphosphate
the incoming nucleoside triphosphate base pairs with its partner on the template strand, it is covalentyl attached to the free 3’ hydroxyl end of the growing DNA strand
new DNA strand synthesized how by what
5’ to 3’ by DNA polymerase
where does energy for polymerization come from
hydrolysis of high energy phosphate bond in incoming nucleoside triphosphate and release of pyrophosphate
polymerase adds incorrect nucleotide, what happens
mispaired nucleotide removed by proofreading, correcly paired3’ end allows additon of next nucleotide
steps of rep fork replication
local opening of helix, RNA primer syntheisis, leading strand begins, RNA primers start lagging strand syn, fork continues to open
telomerase extends which end via what
3’ end via its bound RNA template
in telomere, what completes the lagging strand
DNA polymerase a
reaction for a nicked double helix
step 1 - ATP hydrolyzed, AMP placed on 5’ phosphate, other strand binds to the AMP
step 2 - AMP released, makes continuous DNA strand
deamination changes what to what
C to U
deamination results in what after replication
one sequence is unchanged, one is mutated to where a G has been changed to an A
depurination does what
removes A
depurination results in what after replicaiton
one sequence unchanged, another single base deletion where AT nuc was deleted
describe base excision repair
uracil NDA glycosylase removes deaminated nuc, AP endonuclease adn phosphodiesterase remove sugar phosphate, DNA polymerase adds new nucleotide using bottm strand as template, DNA ligase seals the break
describe nucleotide excison repaur
excision nuclease finds site around damage to cut, helicase removes where the nuclease cut (12 nt gap), DNA polymerase adds new nucleotides using bottom strand as template, ligase seals break
probes labeled by synthesizing them with what conjugated to what
nucleotides conjugated to fluorophoes
DNA primase does not require what
base paired 3’ end as a starting point
lagging strand rep steps
new rna prmer syn by primase, DNA poly adds nucleotides to 3’ end of new RNA primer to syn okazaki frag, poly finishes okazaki frag, prev RNA primer removed by nucleases and replaced with DNA, nick sealed by ligase
For which cell type(s) in your body is the deletion of telomerase inconsequential?
cells that are dying frequenctly or being replaced
UV radiation causes what, explain
causes pyrimidine dimer formation, two pyrimidines fuse together
differences in bacterial vs human nucleotide excision
bacteria - 12 nt gap
human - helicase unwinds DNA duplex, excision leaves 30 nt gap
Hae iii results in what
blunt ends
Eco RI results in what
sticky ends
Hindiii results inw ath
sticky ends
Question 1: Which of the following is a palindromic restriction site (all written 5’ to 3’) that would appear most frequently in a bacterial genome with 60% GC content?
A) CCCAATTCCC B) CGCTATAGCG C) GGCCGGCC D) CCATGG
E) GGAAGG
D
plamid and DNA fragment are cut by what
same restriction enzymes
Ifweusethesamerestrictionenzyme to cut both plasmid and the fragment, what other reaction product can we get from the Ligation reaction?
self ligated plasmid,
ligation of fragments with staggered overhangs reaciton
2 sticky ends + ligase and ATP = joined
conversion of staggered to blunt ends what happens
staggered end filled in by DNA poly + dNTPs; ligase adn ATP join the two
first cycle of amplificaiton describe it
heat to separate strands, cool to anneal primers, DNA syntheisis
what does pcr do
amplifies target region exponentially, doubel stranded
explain generation of labeled DNA probes
start with purified fragment of duplex DNA, denature and anneal with mix of hexanucletides, add DNA polymerase and 32P labeled nucleotides, DNA polymerase incorporates 32p nucleotides, resulting in a population of readiolabeled DNA molecules that contain seq from both strands
Question 2: The Stokes Shift explains the phenomenon of why a fluorophore will absorb photons at a shorter wavelength than it emits them.
A) True B) False
true
difference between ddntp and dntps
ddntp has 3’ H that prevents strand extension at 3’ end
process of DNA sequencing
start with single strand DNA fragment to be sequenced, add primer, add small amounts of
LABELED ddntps and excess amounts of unlabeled dntps, gives you mix of DNA products each containing a chain terminatiing ddntp labeled with specific flurorscent marker
How many copies of each gene does a diploid (2n)
human cell have?
2
You are testing a new method of detecting trisomy 21 in gametes via FISH.
What would you design your DNA probe to target?
I would want to target a sequence that is specific to chromosome 21
you are testing a new method of detecting trisomy 21 in gametes via fish
How would you control for the non-specific binding of your probes?
-ensure the chosen sequence is highly specific to chromosome 21,
- use two different probes each labeled with different phlorophore, for different chromosomes, can help confirm that we actually targeted chromosome 21
You are testing a new method of detecting trisomy 21 in gametes via FISH…
What are the expected FISH results if a metaphase cell you are testing is positive
or negative for trisomy 21?
if negative - there will be two signals on chromosome 21, since there is just 2 copies of it
if positive - there will be 3 signals on chromosome 21 bc there are 3 copies of it
Fluorophores absorb photons at a what wavelength to achieve excitation
shorter wavelength
florophores emit photons at what wavelength
longer wavelength
what is the mechanism for the repair
of depurinated nucleotides? why
base excision repair (ap endonuclease will recognize the site as a missing purine)
What strategy could we use to ensure
get a higher frequency of recombinant
DNA ligations?
inc conc of plasmid and fragment
: What is the minimum number of ATP molecules
required to for DNA Ligase to ligate a single DNA fragment
cut with EcoRI into a single plasmid also cut with EcoRI?
2, one on each DNA end
How can we ensure that only
bacteria that contain our plasmid of
interest are replicated?
use an antibiotic selection marker (resisitance gene) which allow the bacteria that took up recombinant DNA to survive and kills all the others
