15 Questions
What is the purpose of DNA primer constructs in a PCR experiment?
To function as primers in PCR reactions
In PCR, what is the role of heat-resistant DNA polymerase?
To replicate the DNA sequence of interest
What is the purpose of denaturing the DNA in the PCR cycle?
To separate the double-stranded target DNA
What is the significance of annealing of primers to single-stranded DNA in PCR?
To function as primers in PCR reactions
What is the purpose of having two DNA primer constructs for a PCR experiment?
To bracket the DNA sequence of interest for amplification
What is required for a PCR experiment to make copies of a specific segment of DNA?
Thermocycler
What is the function of flanking sequences in a PCR experiment?
To bracket the DNA sequence of interest
What is the purpose of adding DNA polymerase and deoxyribonucleoside triphosphates in excess to the PCR mixture?
To initiate the synthesis of two new chains complementary to the original DNA chains
Which end of the primer does DNA polymerase add nucleotides to during the chain extension process in PCR?
3′-hydroxyl end
Why is a heat-stable DNA polymerase (e.g. Taq polymerase) used in PCR?
To prevent denaturation of the polymerase at high temperatures
What is the outcome of running 20–30 cycles during the PCR process?
Amplifying the DNA by a million-fold to a billion-fold
What is the role of Taq antibody in the PCR process?
To stop the reaction
Where was Thermus aquaticus, the bacterium species used for heat-stable DNA polymerase, discovered?
Yellowstone National Park, USA
Who discovered the bacterium species Thermus aquaticus?
Thomas Brock
What is added at each successive cycle in PCR due to using a heat-stable DNA polymerase?
Nothing is added at each successive cycle
Study Notes
PCR Experiment
- DNA primer constructs are used to initiate PCR amplification of a specific segment of DNA.
- Heat-resistant DNA polymerase (e.g. Taq polymerase) is used to synthesize new DNA strands in high-temperature PCR cycles.
DNA Denaturation
- Denaturing the DNA in the PCR cycle melts double-stranded DNA into single-stranded DNA, allowing primers to anneal.
Primer Annealing
- The purpose of annealing primers to single-stranded DNA is to provide a template for DNA synthesis.
- Two DNA primer constructs are used to bind to opposite strands of the target DNA segment, enabling amplification.
PCR Requirements
- To make copies of a specific segment of DNA, a PCR experiment requires DNA polymerase, deoxyribonucleoside triphosphates, and primers that flank the target sequence.
Flanking Sequences
- Flanking sequences are the regions of DNA adjacent to the target sequence, which are amplified along with the target sequence.
PCR Reaction Mixture
- Adding DNA polymerase and deoxyribonucleoside triphosphates in excess to the PCR mixture ensures efficient DNA synthesis.
Chain Extension
- During the chain extension process, DNA polymerase adds nucleotides to the 3' end of the primer.
Heat-Stable DNA Polymerase
- A heat-stable DNA polymerase (e.g. Taq polymerase) is used in PCR to withstand the high temperatures required for DNA denaturation.
PCR Cycles
- Running 20-30 cycles during the PCR process results in exponential amplification of the target DNA sequence.
Taq Antibody
- There is no role for Taq antibody in the PCR process (Taq refers to the bacterium Thermus aquaticus, which is the source of the heat-stable DNA polymerase).
Thermus Aquaticus
- Thermus aquaticus, the bacterium species used for heat-stable DNA polymerase, was discovered in hot springs in Yellowstone National Park.
- The bacterium was discovered by Thomas Brock and Hudson Freeze in 1969.
PCR Amplification
- At each successive cycle, the number of amplified DNA molecules doubles due to the use of a heat-stable DNA polymerase.
Test your knowledge on Gel Electrophoresis, Southern Blotting, and Polymerase Chain Reaction (PCR) techniques used in DNA extraction and analysis. Learn about the importance and applications of these methods.
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