PCR and electrophoresis Flashcards
What is PCR? (3)
PCR is an in vitro gene cloning technique;
Copies DNA outside of a living organism;
Can produce billions of copies in a few hours
What is PCR used in? (3)
Forensic science;
Evolutionary biology;
Diagnostics
What is an advantage of PCR? (1)
Works with very small samples
What is a disadvantage of PCR? (1)
Increased chance of contamination
How are DNA fragments amplified using in-vitro cloning (PCR)? (5)
Reaction mix: DNA sample, free nucleotides, primers, DNA polymerase;
Heat to 95°C: Hydrogen bonds break, DNA strands separate;
Cool to 50-65°C: Primers anneal to DNA strands;
Heat to 72°C: DNA polymerase adds complementary nucleotides;
Repeat cycle: DNA doubles each cycle (exponential amplification)
Why are primers needed in PCR? (2)
Allow DNA polymerase to attach to the DNA strands;
Two different primers are required for the different sequences at the start and end of the target DNA
What happens in each PCR cycle? (2)
After each cycle, the number of DNA fragments doubles;
E.g., after 1st cycle: 4 fragments, after 2nd cycle: 8 fragments
Why does the amount of DNA produced level off in PCR? (2)
Nucleotides and primers get used up;
Without these reagents, no more complementary strands can be synthesised
What are the key differences between PCR and transcription? (4)
Enzyme used: Transcription uses RNA polymerase, PCR uses DNA polymerase;
Nucleotides: Transcription uses RNA nucleotides (uracil), PCR uses DNA nucleotides;
Template: Transcription uses one template strand, PCR uses both strands;
Initiation: Transcription uses start/stop codons, PCR uses primers
What are the differences between PCR and semi-conservative replication? (3)
Length of DNA: PCR replicates short fragments; semi-conservative replication replicates entire DNA;
Strand separation: PCR uses 95°C heat to separate strands; semi-conservative replication uses DNA helicase;
Primers: PCR requires primers, semi-conservative replication does not
What does electrophoresis separate? (2)
Separates DNA, RNA fragments, or proteins; Based on size
What is the first step in electrophoresis of DNA fragments? (2)
Prepare the gel by pouring agarose gel into a tray;
Creating wells at one end
How do you set up the gel for electrophoresis? (1)
Place the gel in a box or tank with the wells near the negative electrode
What is the purpose of adding a buffer solution in electrophoresis? (1)
Add buffer solution to cover the gel to maintain the pH and conduct electricity
Why is loading dye added to DNA samples during electrophoresis? (2)
For visibility;
To help the samples sink into the wells
How are DNA samples added to the gel during electrophoresis? (1)
Use a micropipette to add DNA samples to the wells in the agarose gel
How is a current applied to the gel in electrophoresis? (2)
Connect the box to the power supply;
To run an electrical current through the gel
How does DNA move in electrophoresis? (2)
DNA, being negatively charged;
Moves towards the positive electrode under the influence of the electric current
How do DNA fragments separate during electrophoresis? (2)
Smaller DNA fragments move faster through the gel;
Separating based on size
How are DNA bands visualised after electrophoresis? (1)
Using a staining solution that binds to the DNA
What is electrophoresis of proteins used for? (1)
Identifying proteins in urine or blood samples for disease diagnosis
