DNA Replication Flashcards
What does PCR stand for and what does it mean?
Polymerase Chain Reaction, = synthesis of
DNA by thermostable
DNA polymerase in the
test tube
Define DNA Cloning
Prior to the 1980’s, the primary method for producing many copies of a gene
What is the key element of PCR?
Heat. DNA is submitting through heating and cooling cycles.
Explain the steps of PCR and how it works
Denaturation, Annealing, and Primer Extension. GATHER, BOUND, POLYMERASE, TO MIMIC THE PROCESS OF DNA REPLICATION First, a small amount of DNA is gathered, a pair of primers are used to bind each end of the target sequence, Then a dna polymerase is used, then Four dNTPs (i.e., dATP, dCTP, dGTP, dTTP), and last a few essential ions and salts. They use those ingredients to to mimic the natural dna replication process. A thermocycler is used with cool and heat.
What is a Thermocycler?
A machine that jump-starts each stage of the reaction by raising and lowering the temperature of the chemical components at specific times and for a preset number of cycles.
What Denatursation?
The melting of DNA into individual strands. The started solution is heated to break the bonds joining the strands of a DNA double helix enabling it to separate into two strands.
What is Annealing?
The reaction mixture is quickly cooled, usually between 30 and 65 degrees. This gives the primers an opportunity to bind or anneal to their complementary sequences on the single strands of DNA
What is Extension?
The sample is heated and the DNA polymerase begins to make a new DNA strand by attaching to the primers
What is (RT-PCR)?
Reverse Transcription Polymerase Chain Reaction, this combines real time PCR and reverse transcription
What is Reverse Transcription?
The process that makes dna from rna
What is qPCR?
quantitative real-time PCR (qPCR)
What is PCR used for?
Sequencing, cloning, and analysis in the case of forensics
What are the two primers called?
Forward and Reverse primers
What can we tell if both primers attach to the same piece of DNA and synthesize in the same direction?
That there is no PCR product
Explain the process of replication
Template strands separate, New nucleotides are added according to the templates, and they are joined into the new strand
T/F Replication occurs prior to cell division
True
T/F Replication happens during the G1 phase of the cell cycle
False. It happens during the S phase.
T/F The cell divides producing
two daughter cells during the
mitotic phase
True
In the cell, splitting is performed by a protein called what
Helicase
What temperature does the helicase unwind the DNA helix at?
37 degrees which is body temperature
What are origins of replication?
The origin is a specific sequence in the DNA that is recognized
by DNA polymerase as a start point for replication
The origin is usually an AT rich region, because
AT regions are easier to break.
T/F There is only one
origin of replication
in a single
chromosome
False. There are many origins in a single chromosome.
T/F Each strand can provide a new template for building a new strand
True
T/F New nucleotides are arranged in the
new strand by their ability to
complement the template strand bases
True!
T/F G’s bind with A’s
* A’s bind with C’s
False -
G’s bind with C’s
* A’s bind with T’s
T/F DNA polymerase adds about 1000-5000 bp/minute depending
on conditions
True
T/F Replication can occur in many directions
False. It can only occur in one direction
DNA polymerase can start a new strand by itself
False. it cannot start a new strand by itself. It only can add to an existing strand
What is primase?
An enzyme consisting of RNA is added to a sequence
What is a primer?
In the cell, a short primer sequence consisting of RNA is added
by a enzyme called Primase.
T/F Since the primer is actually made of RNA nucleotides, it must
be eventually removed and replaced with DNA.
True!
T/F The leading strand has a primer at the beginning, and
replication occurs continuously as the DNA unwinds.
True!
T/F The lagging strand grows in the same direction from the
movement of helicase through the production of short segments
of DNA (Okazaki fragments).
False. It is the opposite direction.
T/F The enzyme ligase seals the Okazaki fragments together after
removal of the primers.
True
T/F Before editing the error rate is about 1/100,000.
True
After editing the error rate in replication is about 1/1,000,000
False! It’s After editing the error rate in replication is about 1/10,000,000
T/F These errors create mutations in the DNA from one
generation to the next
True!
Are Mutations in the DNA the source of variation in living
organisms?
YES!
What are the four components required for PCR?
Template DNA (to be copied). (primer, polymerase, dntp, thermocycler)
2. Primers (to initiate the reaction)
3. DNA polymerase (to add the bases)
4. dNTP (deoxyribonucleoside triphosphates) or the A’s, T’s,
C’s, and G’s
5. While PCR can be done with heat plates and ice baths, all
labs now simply use Thermal Cyclers.
What is the temperature for Denature?
94 degrees
What is the temperature for Anneal?
30-65 degres, it’s more variable
What is the temperature for extension ?
72 degrees
Why do our cells require helicase, but we can get by without it
during PCR?
Our cells require helicase instead of denaturation because if we were to heat our
bodies up to 94-95 C, we would die. Plain and simple. That sort of extreme heat
would cause all of our systems to shut down, and cells to commit to apoptosis (cell
death). With PCR we are allowed a certain leniency with environmental controls.
Because of this, we can go through cycles of denaturing and annealing the DNA in
order to better control our product yield. Furthermore, with PCR, you can increase
and decrease the temperature to allow for multiple reactions to occur. However, with
helicase, once you add the enzyme to the PCR reaction, it will unzip all strands and
keep doing so – even the strands we want to keep together.
T/F Amplification always occurs in the 5’ 3’ direction!!
True
If you start with 1 double-stranded DNA molecule as a template and
conduct a PCR for 5 thermal cycles, how many DNA molecules of the
PCR product will you synthesize at the end of the reaction?
- If starting with 1 molecule, the final
number of double stranded DNA molecules can be calculated using 2n
1 double-stranded DNA = 2n = 25 = 32
What side does a primer need to bond onto DNA?
The 3 side, never the 5
