You are ready to start your PCR. You have your genomic template DNA and your designed primers. What
else do you need in your PCR reaction and why?
You need nucleotides to serve as your building blocks for DNA synthesis. You need DNA polymerase to catalyze the addition of building blocks to the replicated strand of DNA.
restriction enzymes recognize specific nucleotide sequences in target DNA
true
Restriction enzymes were originally isolated from bacteria.
true
Restriction enzymes recognize specific DNA sequence sites, and may give products with sticky ends or blunt end products after cleavage
true
Restriction enzymes cleave only the hydrogen bonds between the nucleic acid polymers
false
enzymes that cleave DNA at specific nucleotide
sequences. Because these enzymes function to restrict the transfer of DNA between strains of bacteria, they were called
restriction enzymes
or restriction nucleases
Different bacterial species produce different restriction enzymes, each cutting at a different, specific nucleotide sequence.
The bacteria’s own DNA is protected from cleavage by chemical modification of these specific sequences
true
for a given sample of DNA, a particular restriction enzyme will reliably generate
the same set of DNA fragments
3 steps of PCR
1. heat to separate strands
2. cool to anneal primers
3. DNA synthesis
+DNA polymerase (only 1x sometimes bc heat stable)
+dATP, +dGTP, +dCTP, +dTTP
first products of cycle --> repeat
What are the consequences for a dideoxy DNA sequencing reaction if the ratio of dideoxyribonucleoside triphosphates to deoxyribonucleoside triphosphates is increased? What happens if this ratio is decreased?
If the ratio of dideoxyribonucleoside triphosphates to deoxyribonucleoside triphosphates is increased, DNA polymerization will be terminated more frequently
and thus shorter DNA strands will be produced.
Such conditions are favorable for determining nucleotide sequences that are close to the DNA primer used in the reaction. In contrast, decreasing the ratio of dideoxyribonucleoside triphosphates to deoxyribonucleoside triphosphates will produce longer DNA fragments, thus allowing one to determine nucleotide sequences more distant from the primer
Restriction enzymes cut DNA at specific sites that are always located between genes.
False.
Restriction sites are found at random throughout
D. PCR utilizes a heat-stable DNA polymerase because for each amplification step, double-stranded DNA must be heat-denatured.
True. Each polymerization reaction produces double stranded DNA that must, at each cycle, be denatured to allow new primers to hybridize so that the DNA strand can be copied again.
The energy for the polymerization reaction comes from
the hydrolysis of a high-energy phosphate bond in the incoming nucleoside triphosphate and the release of pyrophosphate
results in an amino acid substitution that alters protein function
C) Missense mutations
mutation(s) with no impact on protein function
silent
neutral
give an example of a
Small genetic changes (base pair changes, gain of genes) can be advantageous/beneficial
Peppered moths are nocturnal and spend their days dozing on tree trunks or walls, so this new colouring gave them a much better chance of hiding from hungry birds in the smoke-stained world of industrial England
Mutation occurs in non-coding regions of DNA
silent
neutral
Mutation occurs in gene but does although it changes the amino acid sequence, the properties of the new amino acid do not change protein function
D) neutral
Deamination
C now read as U
Unrepaired deamination can lead to
amino acid substitution or a nonsense mutation (stop codon created to make a truncated protein
Unrepaired depurination will cause
replication machinery to “skip” a nucleotide and shift how all the remaining information will be read during translation.
frame shift
Spontaneous DNA damage
depurination and deamination
Excision Repair is used when [one/two] strand(s) of the helix has mutations
one
Types of damage corrected by excision repair
a.Spontaneous DNA damage
b.Induced DNA damage
c.mismatch repair targets incorrect Watson-Crick base pairs
3 different types of DNA excision repair
NER
BER
mismatch repair
The repair DNA polymerase:
binds 3’ OH end of the DNA strand and adds new nucleotides in the 5’ - 3’ direction
has proofreading abilities.
is different than the DNA replication polymerase but the same repair polymerase can be used for all types of excision repairs.
The ligase used in excision repair is the same type used
to seal the nicks between the Okazaki fragments during DNA replication
Mechanism to repair double strand breaks caused by ionizing radiation and oxidation
nonhomologous end-joining
homologous end-joining
Mechanism to repair double strand breaks caused by ionizing radiation and oxidation
to seal the nicks between the Okazaki fragments during DNA replication
Preferred method but can only be used during short window of cell cycle after
DNA replication has occurred, but before the chromosome copies have been separated in M-phase
Homologous recombination
in repair of double stranded
DNA breaks
PCR is used to
Amplifying gene to study it or the product of it
Detection of bacteria or viruses – e.g. Covid PCR test
Studying and mapping genomes (finding the sequence of a genome and identifying genes)
Diagnosis of genetic disorders
What do we need to replicate DNA? pcr
1.Template sequence
2.A way to separate the double stranded template DNA
Heat takes the place of helicase
3.Primers
We use synthesized DNA primers, so we eliminate the need for primase, nuclease, and ligase
