PCR Flashcards
What is PCR?
Technique to make many copies of a specific DNA region in vitro
What does PCR require?
Thermostable DNA polymerase Taq polymerase
DNA primers designed specifically for the DNA region of interest
Nucleotides
Template DNA
When is PCR used?
DNA cloning
Medical diagnostics
Forensic analysis of DNA
What is the goal of PCR?
Make enough of the target DNA region that it can be analyzed or used in some other way
DNA may be
- sent for sequencing
- visualised by gel electrophoresis
- cloned into a plasmid for further experiments
What is Taq polymerase?
PCR requires DNA polymerase enzyme that makes new strands of DNA, using existing strands as templated
DNA polymerase typically used in PCR is Taq polymerase
After the heat-tolerant bacterium from which it was isolated (Thermus aquaticus)
When is Taq polymerase most active?
70 degrees
What are primers?
Short sequences of nucleotides that provides a starting point for DNA synthesis
Experimenter determines the region of DNA that will be copied by the primers they choose
What is the average length of primers?
Around 20 nucleotides in length
How many primers are used in a PCR reaction?
Two primers are used in each PCR reaction
They are designed so that they flank the target region
Given sequences that will make them bind to opposite strands of the template DNA
Just at the edges of the region to be copied
What are the steps of PCR?
- Ingredients are assembled in a tube, along with cofactors needed by the enzyme
- Denaturation
- Annealing
- Extension
What temperature does denaturation happen?
96°C
What happens during denaturation?
Heat the reaction strongly to separate or denature the DNA strands
Provides a single-stranded template for the next step
What temperature does annealing happen?
55 - 65°C
What happens during annealing?
Cool the reaction
So the primers can bind to their complementary sequences
On the complementary sequences on the single-stranded template DNA
What temperature does extension happen?
72°C
What happens during extension?
Raise the temperature so Taq polymerase extends the primers
Synthesizes new strands of DNA
How many times is this process repeated?
Cycle repeats 25-35 times in a typical PCR reaction
How many hours does a normal PCR take?
2-4 hours
What enables the rapid growth of the DNA?
It’s not just the original DNA that’s used as a template each time
The new DNA that’s made in one round can serve as a template in the next round of DNA synthesis
Many copies of the primers and Taq polymerase floating around in the reaction
Number of DNA molecules can roughly double in each round of cycling
How long does annealing take?
Around 1 minute
Determined by the binding between the bases
A and T - weaker bonds, the melting temperature is lower
C and G - stronger bonds, the melting temperature is higher
What is a thermocycler machine?
Performs PCR automatically
How can PCR be used to diagnose UTIs?
All eubacteria have 16s ribosomal gene that codes for 16s ribosomal DNA
Design primers that stick to the constant region on the constant DNA that codes for the 16s gene
Metagenomic sequencing
What is metagenomic sequences used for?
Strain identification
Mutation
Look for genes for antibiotic resistance
What is the advantage of PCR?
Only need small amount
What are the disadvantages of PCR?
Need to know the sequence you are investigating
Expensive
Takes time
