genetics chapter 7 part 2 Flashcards
telomerase is a what?
an RNA-dependent DNA polymerase that carries its own RNA template to make new dna sequences
what do most telomeres contain?
a knotted fold, or T loop
what are e.coli daughter dna strands synthesized by?
dna pol iii holoenzyme
holoenzyme
a multiprotein complex where pol iii is associated with the additional components needed for full function
is holoenzyme or replisome bigger?
replisome
how does dna polymerase elongate stands of dna?
by adding nucleotides to the 3’ end of a pre-existing strand/primer
what are rna primers synthesized by?
primase
what has the most fully characterized origin of replication?
sacchromyces cerevisiae (yeast)
what are multiple origins or replication called?
autonomously replicating sequence (ARS)
topoisomerase
catalyze controlled cleavage and rejoining of the dna that prevents over winding and releases torisonal stress
what does DnaA do?
binds to the 9-mer sequences, bends DNA, and breaks hydrogen bonds in A-T rich sequences of the 12-mer region
what is DnaB carried by?
DnaC
what does dnaC carry?
DnaB
what is DnaB?
a helicase that uses ATP energy to break hydrogen bonds of complementary bases to seperate the strands and unwind the helix
what keeps the unwinded strands from reannealing?
SSB (single stranded binding protein)
what eases a supercoiled DNA?
topoisomerase
where is the replisome found?
at each replication fork
how many dna pol iii does the replisome carry?
2, one for leading strand, one for lagging strand
what 3 activities does DNA pol i use?
5’-3’ exonuclease activity to remove the primers
5’-3’ polymerase activity which adds DNA nucleotides to the 3’ end of the preceding DNA segment
what does DNA ligase do?
it glues together the okazaki fragments by creating phosphodiester bonds
how many protein subunits does DNA holoenzyme carry?
11 and 2 pol iii core polymerases
what are the 2 pol iii core polymerases in the DNA holoenzyme tethered to?
Tau protein
what are the tau proteins joined together by?
a clamp loader where 2 additional proteins from the sliding clamp
why is dna syntheses very accurate?
bc dna polymerases have dna proofreading ability
dna polymerase enzymes have what type of activity? ?????
3’-5’ EXOnuclease activity (reverse synethesis direction)
what happens when there is a DNA mismatch?
inability of the mismatched bases to form the appropriate h-bonds
how is replication in telomeres?
leading strand is replicated all the way to the end while the lagging strands need for a primer means that they cannot be completely replicated???
what are telomeres?
repetiive sequences at the end of the chromosome
what do telomeres making sure doesnt happen?
makes sure vital genes aren’t affected by incomplete chromosomes
what does the T loop in the telomere bind to?
the shelterin protein complex
what does the shelterin protein complex do?
it protects the telomeres from degradation
what does telomere shortening eventually trigger?
apoptosis
what is the hayflick limit?
this represents the number of cell cycles before apoptosis occurs
what is werner syndrome?
early onset aging due to RECQL2 gene encoding a helicase required from telomerase activity
what is dyskeratosis congenita?
a disorder associated with loss of function of a gene- DKC1 - this gene is supposed to encode a protein needed for normal telomerase function
what cells is telomerase normally turned off in?
somatic cells
What is the most common mutation in cancers of all types?
TERT reactivation (this encodes the reverse transcriptase function of a telomerase)
what does PCR stand for?
polymerase chain reaction
what is PCR?
an automated version of DNA replication that produces millions of copies of a short target DNA segment
what does PCR require?
-a double stranded DNA template
-the four dna nucleotides
-a heat stable DNA polymerase
-two different single-stranded dna primers
-a buffer solution
what is the most used dna polymerase?
taq
what is taq isolated from
thermus aquaticus
what provides the starting point for Taq in PCR?
two DNA sequences called PCR Primers
what are the three steps of PCR?
-denaturation- heated to 95ºC (to denature protein)
-annealing: temp reduced to 45-68º C (allows primer to bind to their complementary sequences in the target DNA)
-extension: temp raised to 72ºC (allows Taq polymerase to synthesize DNA)
what is qPCR?
a modification to traditional PCR where a spectrophotometer is used to measure DNA production in real time
what is the most common detection method for qPCR?
SYBR Green
what are the benefits of qPCR
-can be used to quantitate the starting amount of template DNA accurately
-reduces detection time
-does not require electrophoresis
what is ddPCR?
the newest version of PCR
that creates thousands of tiny droplets each containing all the components of a PCR reaction
PCR is preformed in each drop
amount of dna per drop is read
results are analyzed
what are the benefits of ddPCR?
highly quantitative and very sensitive
very good percision
what are dNTPs
the four standard deoxynucleotide bases
what is dideoxy sequencing?
it uses DNA polymerase to replicate new DNA from a single-stranded template
what is a ddNTP
dideoxynucleotide (ddNTP)
when does replication cease in sanger sequencing?
when a ddNTP is incorporated onto the product DNA molecule
what are automated dna sequencers?
it uses a single reaction for each DNA sequence where all 4 ddNTPs are included
how do they tell the ddNTPs apart?
with a fluorescent marker
what does the florescent marker do?
they each have a different wavelength and a laser light excites the florescent tag on each fragment as it passes. the wavelength is read and information is recorded by a computer
how do third generation mothods and Next Generation Sequencing differ from sanger sequencing?
- tgs and ngs methods use PCR to amplify DNA, this allows feasible sequences of repetitive DNA
- tgs and ngs are “massively parallel” this means that millions of sequencing reads may be done in each sequencing run
