PCR Flashcards
Describe PCR
A method used for isolating specific pieces of DNA of an already known gene amplification of DNA from a small amount of template DNA without using a living organism
- Invented by Ka y Mullis( Nobel Prize 1993)
- Widely regarded as one of the most important inventions in molecular biology in the 20th century
What is DNA Amplification?
Facilitates DNA identification and manipulation
- Detection of infectious organisms, examples: bacteria(tuberculosis), viruses (AIDS, HIV)
- detection of genetic variations such as mutations
Summarize the steps of PCR
Repeated 30-40 times
Step 1: denaturation
Step 2: annealing of primers
Step 3: extension/synthesis
Describe the steps of PCR
First, the genetic material is denatured, converting the double stranded DNA molecules to single strands
The primers are then annealed to the complementary regions of the single stranded molecules
In the third step, they are extended by the action of the DNA polymerase
-All the steps are temperature sensitive and the common choice 9f temperatures are approximately 94 C, 60C and 70C respectively
What makes good primers?
Short ( usually 17-25 nucleotides) single stranded DNA molecules that bind to specific DNA sequences and act as initiation sites for DNA polymerization
This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature
Describe a bad primer
Poor primers could result in little or even no PCR product
Alternatively, poor primers could amplify many unwanted DNA fragments
Therefore, it is crucial that you design your primers carefully
Primer design requires extensive computer-based sequence analysis
Ideally primers would be complementary only to the target sequence
This would ensure that the taq polymerase only copies the target region
What is the Tm?
The Tm is the temperature at which one half of DNA duplexes in a sample will dissociate and become single
At temperatures above the Tm the DNA molecules will be in the single stranded form; at temperatures below the Tm the DNA can form the double stranded form
The annealing temperature for a PCR reaction is based on the melting temperature (Tm) of the primers
How does melting temperatures affect primers?
Primers with melting temperatuures in the range of 52-58 C generally produce the best results. Primers with melting temperatures above 65 degrees Celsius have a tendency for secondary annealing
Typically, the annealing reaction is carried out about 5 degrees Celsius below the melting temperature
-If the annealing temperature is too high, the primer will not anneal to the target DNA
If the annealing temperature is too low…
The primer will mix-anneal to sequences which aren’t perfectly complementary
The most important consequence of this is that the two primers designed for a PCR experiment should have very similar Tm
- Typically the Tm should within 5 degrees Celsius of each other. The closet the Tms the better
- The Tm should be within 5 degrees C each other. The closer the Tms the better
- The Tm of a DNA molecule is dependent on its sequence, however the relationship between sequence and Tm is not simple. In general, the greater the GC content of DNA the higher its Tm
How can we calculate Tm?
- Wallace rule
Tm(in Celsius)= 2(A+T)+4(C+G)
(A+T)= sum of C and G residues in the pr8mer (C+G)= sum of C and G residues in the pr8mer
- DNA calculator
This website will allow you to fill in your primer sequence to determine the length, Tm, and molecular weight for your primers and if there will be any secondary structures or possibilities of primer dimers
What are hairpins?
A primer that is base pairing with itself cannot base pair with its target DNA
Primers must be designed to minimize intra molecular base pairing. Intra molecular base pairing is usually analyzed using computer programs
Avoid primers that contain more than a string of 3 intra molecular base pairs
What is Hererodimer formation?
Primers can also participate in intermolecular base pairing.
This is base pairing between two different primer molecules
If the base pairing is between the forward a;d the reverse primer it 8s called heterodimer formation
Describe self-dimer formation
If the base pairing is between just one of the two primers it is called self-dimers formation
The example primer used above can form several self-dimers
Both the given examples are problematic
The first is highly stable structure with numerous base pairs
If the primers are base pairing with themselves they cannot base pair with the target DNA
