Primer Design Flashcards
What are primers?
They are a component of typical CPR mix.
What are the two main types of primers?
Forward and reverse primers
Forward and reverse primers are a pair of __________ that __________ with the DNA template and __________.
Short DNA fragments; hybridize; defines the region that will be amplified
What is the primer?
CO1/COX1/MTCO1 gene (Cytochrome oxidase subunit 1)
This region is not prone to mutations and nucleotide substitutions.
Conserved region
This is where the primer attaches to a DNA template and targeted region gene sequence.
Primer annealing
What is the common annealing temperature?
45ºC - 60ºC
(it differs according to design primer, though)
What are the 7 criteria for checking primer efficiency?
- Primer length
- Primer melting temperature
- Primer annealing temperature
- GC Content and GC Clamp
- Secondary structures
- Repeats and runs
- Cross homology
What is the ideal primer length?
15-22 of 18-24 number of bases
This is the temperature at which 50% of the primer and its complement are hybridized.
Primer Melting Temperature
What is the ideal melting temperature?
52ºC - 58ºC
Max: 65ºC
What is the formula for checking the primer melting temperature (Tm)?
Tm = 2(A+T) + 4(G+C)
What happens when the primer melting temperature is too high?
Secondary annealing
What happens when the primer melting temperature is too low?
Unstable and reduced binding
This is the optimal temperature in which the primers bind to the target sequence.
Primer annealing temperature
How do you solve for the primer annealing temperature?
Melting temperature - 5ºC
What is the ideal GC Content?
40% - 60%
The GC clamp increases specificity by selecting a primer sequence with G or C bases within the last __________ bases from the __________ end of the primers.
5; 3’
Why is it particularly GC content and clamp, not AT?
GC nucleotide bases have 3 hydrogen bonds, making them more secure compared to AT bonds with only 2 hydrogen bonds
What is the maximum repeats and runs?
4 for both
This avoids a di-nucleotide from occurring several times consecutively.
Repeats
This avoids a long run of single bases.
Runs
This type of amplification yields different bands of different sizes. How do you interpret such result?
Non-specific amplification; Poorly designed
This type of amplification yields a single band. How do you interpret such result?
Specific Amplification; Ideal result
What are the three types of secondary structures?
- Hairpin
- Self-dimer
- Cross-dimer
This type of secondary structure holds intramolecular interactions within the primer.
Hairpin
This type of secondary structure holds intramolecular interactions between the same sense primers.
Self-dimer
This type of secondary structure holds intramolecular interactions between the sense and antisense primers.
Cross-dimer
This type of secondary structure is where forward and reverse primers bind to each other instead of the targeted primer region.
Cross-dimer
