Module 7 Flashcards
What is the central dogma?
DNA replication > transcription > translation
T/F Genetic information flows from DNA to RNA to Protein
TRUE
Each parental strand serves as a template for formation of what?
antiparallel, complementary
daughter strand
Completion of replication results in the formation of what?
two identical daughter
duplexes
Define semi-conservative
What direction is DNA synthesized?
one parental and one
daughter strand
5’ to 3’
DNA replication is most often ___________, proceeding in
both directions from each origin of replication, forming a what?
bidirectional
replication bubble
Bacterial chromosomes have how many origins of replication?
Eukaryotic?
a single origin
MULTIPLE origins
why is it logical to have an AT
rich region here?
fewer hydrogen bonds, making it
easier to separate
What binds at the origin and opens the DNA double helix in both directions away from the origin?
helicase
Why do Single-strand binding proteins (ssb) bind the
separated DNA strands?
to prevent them from reannealing
Unwinding of circular chromosomes creates stress and can lead to?
Which enzyme catalyze controlled cleavage and rejoining of DNA that prevents over-winding?
supercoiled DNA
topoisomerase
What builds new strands
5ʹ-3ʹ?
What replaces RNA
primers with DNA?
DNA pol III
DNA pol I
DNA _____ seals nick at 5ʹ end of
replaced primer
ligase
What other protein involved in DNA
replication ligates DNA?
Topoisomerase cleaves and ligates DNA to
avoid super-coiling
Which protein blocks extension of
replication fork?
Tus protein
EUKARYOTIC
Multiple origins along a (CIRCULAR OR LINEAR) chromosome
T/F Termination occurs at chromosome ends
- ______ __________ epsilon responsible for leading
strand synthesis - DNA polymerase delta (lagging strand*) runs
into ___’ end of adjacent Okazaki fragment, causing
displacement and a single-strand
____________________ cleaves the flap
LINEAR
TRUE
DNA polymerase
Flap endonuclease (FEN1) cleaves the flap
What is the chromosome shape of prokaryotes and eukaryotes?
🦠 Prokaryotes: Circular
🧬 Eukaryotes: Linear
How many origins of replication do prokaryotes and eukaryotes have?
🦠 Prokaryotes: One (singular)
🧬 Eukaryotes: Multiple
What direction does DNA replication occur in both prokaryotes and eukaryotes?
Bidirectional (5’ to 3’)
Where does DNA replication terminate in prokaryotes and eukaryotes?
🦠 Prokaryotes: ter sites (Tus proteins help stop replication)
🧬 Eukaryotes: Chromosome ends
Do prokaryotes need an end-replication solution?
🦠 No, because their DNA is circular
What is the end-replication solution for eukaryotes?
Telomeres
Which enzyme synthesizes the leading strand in prokaryotes and eukaryotes?
🦠 Prokaryotes: DNA pol III
🧬 Eukaryotes: DNA pol epsilon
Which enzyme removes primers in prokaryotes and eukaryotes?
🦠 Prokaryotes: DNA pol I
🧬 Eukaryotes: FEN1
Which enzymes are involved in DNA replication in both prokaryotes and eukaryotes?
✔ Topoisomerase
✔ Helicase
✔ SSB (Single-Strand Binding Proteins)
✔ Primase
✔ Ligase
Match each enzyme with its functions
A. Topoisomerase
B. Helicase
C. SSB (Single-Strand Binding Proteins)
D. Primase
E. Ligase
Seals gaps in DNA
Stabilizes unwound DNA
Unwinds DNA
Relieves supercoiling
Lays down RNA primers
A. Topoisomerase – Relieves supercoiling
B. Helicase – Unwinds DNA
C. SSB (Single-Strand Binding Proteins) – Stabilizes unwound DNA
D. Primase – Lays down RNA primers
E. Ligase – Seals gaps in DNA
Mitochondria have their own _________ genomes
circular
How fast are DNA polymerases in prokaryotes and eukaryotes?
🦠 Prokaryotic (DNA pol III in E. coli): 700-1000 bases/second
🧬 Eukaryotic DNA polymerases: 50-100 bases/second
How often do DNA polymerases make mistakes?
~1 in 100,000 bases
How do DNA polymerases correct mistakes?
3’ → 5’ exonuclease activity (proofreading)
How much does proofreading improve accuracy?
Reduces error rate to ~1 in 100 million bases
How do other repair systems improve accuracy further?
- continuously scan for errors resulting in low error rates
~1 in 10 billion bases (10⁻¹⁰)
What is the final DNA mutation rate per base pair after repair?
1 in 10 billion bases (1 × 10⁻¹⁰/bp)
How often does a mutation occur in humans?
1 mutation per 3 cell divisions
How many novel mutations are in each sperm and egg cell?
Sperm cell: ~124 novel mutations
Egg cell: ~10 novel mutations
How many cell divisions occur before sperm and egg formation?
Sperm: ~400 divisions from zygote
Egg: ~30 divisions from zygote
What is used instead of Helicase, SSB, and topoisomerase to denatures DNA?
heat
20bp single stranded DNA that flanks your target region
Primers
which DNA polymerase is heat resistant
DNA polymerase III
The traditionally used DNA polymerase, Taq polymerase, is isolated from what and found where?
Thermus aquaticus, which is found naturally in hot springs
Which polymerase can handle very very hot temperatures – hotter than what is needed for DNA denaturation?
Taq polymerase (Thermus
aquaticus) 70 -75 Celsius
name the 4 Steps of PCR
- Denaturation: (rxn heated to ~95°C to denature the DNA)
- Primer annealing: (rxn temperature is reduced to ~45–68°C so primers can hybridize)
3.Primer extension: (rxn raised to
~72°C to allow Taq polymerase to synthesize DNA)
- Repeat steps 10-35 times (DNA product increases at ~2n, where n = # of cycles)
1 cycle = _ copies
_ cycles = 4 copies
_ cycles = _ copies
4 cycles = _ copies
1 cycle = 2 copies
2 cycles = 4 copies
3 cycles = 8 copies
4 cycles = 16 copies
Amplified DNA fragments are separated from the rest of the reaction mixture by what?
gel electrophoresis
- DNA is _________ charged, so it travels towards the positive electrode?
- In a gel, smaller (shorter) DNA molecules move slower or faster?
3.What is used to determine DNA
size?
- negatively
- faster
- A ladder
- Variable number tandem repeats (VNTRs/STRS short tandem) are used as what?
- TNRR units up to how many bp in length found throughout the genome?
- a type of genetic marker
- up to 20 bp in length
T or F VNTRs are inherited, and can be detected through PCR amplification
True
Some short tandem repeats can
cause what?
genetic disease (e.g., Friedreich Ataxia, Huntington’s Disease, Fragile X syndrome
The 2 steps to visualize sequence of bases from DNA fragment of interest?
- Dideoxynucleotide DNA sequencing (dideoxy sequencing), aka chain termination PCR?
- (Capillary) electrophoresis of PCR products
Dideoxy sequencing starts with what?
a regular PCR
In addition to regular dNTPs, each reaction contains a small amount of what (dd which lacks a 3¢-OH group)?
one dideoxynucleotide
Whenever a ddNTP is incorporated into the product DNA molecule, replication ceases this is known as what?
“chain termination PCR
What percentage of the human genome codes for proteins?
2%
_________ Strand- Replicated continuously to the end
_________ Strand - Requires primers and can’t fully replicate
_________________ - No room for final lagging strand primer
_____________ - Repetitive sequences that protect genes
Leading
Lagging
End-Replication Problem
Telomeres
What is the repeated telomere sequence in vertebrates?
How many times is the telomere sequence repeated?
Are telomeres conserved across vertebrates?
What remains after replication at the telomere end?
Which strand of telomere DNA has the overhang?
5’- TTAGGG -3’
300-5000 repeats
Yes, they are highly conserved.
A 3’ overhang (~50 bases)
The G-rich strand
What does telomerase use as a template?
An RNA template complementary to the leading strand overhang
What is the function of telomerase?
Extends the leading strand so the lagging strand can be synthesized fully
Why is telomere length important?
chromosome stability, cell longevity, and reproductive success
Where is telomerase active in eukaryotes?
germ-line cells and some stem cells
Where is telomerase inactive?
differentiated somatic cells
What happens if telomerase is reactivated?
can lead to aging cells that continue to proliferate, a feature of many cancers
What is Polymerase Chain Reaction (PCR)?
DNA replication in a tube that makes millions of copies of a short DNA segment.
What can PCR be used for?
Cloning, gene detection, gene analysis, sequencing, and repeat analysis
