Primer Design

Question 1

What are primers?

Answer 1

They are a component of typical CPR mix.

Question 2

What are the two main types of primers?

Answer 2

Forward and reverse primers

Question 3

Forward and reverse primers are a pair of __________ that __________ with the DNA template and __________.

Answer 3

Short DNA fragments; hybridize; defines the region that will be amplified

Question 4

What is the primer?

Answer 4

CO1/COX1/MTCO1 gene (Cytochrome oxidase subunit 1)

Question 5

This region is not prone to mutations and nucleotide substitutions.

Answer 5

Conserved region

Question 6

This is where the primer attaches to a DNA template and targeted region gene sequence.

Answer 6

Primer annealing

Question 7

What is the common annealing temperature?

Answer 7

45ºC - 60ºC

(it differs according to design primer, though)

Question 8

What are the 7 criteria for checking primer efficiency?

Answer 8

1. Primer length
2. Primer melting temperature
3. Primer annealing temperature
4. GC Content and GC Clamp
5. Secondary structures
6. Repeats and runs
7. Cross homology

Question 9

What is the ideal primer length?

Answer 9

15-22 of 18-24 number of bases

Question 10

This is the temperature at which 50% of the primer and its complement are hybridized.

Answer 10

Primer Melting Temperature

Question 11

What is the ideal melting temperature?

Answer 11

52ºC - 58ºC

Max: 65ºC

Question 12

What is the formula for checking the primer melting temperature (Tm)?

Answer 12

Tm = 2(A+T) + 4(G+C)

Question 13

What happens when the primer melting temperature is too high?

Answer 13

Secondary annealing

Question 14

What happens when the primer melting temperature is too low?

Answer 14

Unstable and reduced binding

Question 15

This is the optimal temperature in which the primers bind to the target sequence.

Answer 15

Primer annealing temperature

Question 16

How do you solve for the primer annealing temperature?

Answer 16

Melting temperature - 5ºC

Question 17

What is the ideal GC Content?

Answer 17

40% - 60%

Question 18

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.

Answer 18

5; 3’

Question 19

Why is it particularly GC content and clamp, not AT?

Answer 19

GC nucleotide bases have 3 hydrogen bonds, making them more secure compared to AT bonds with only 2 hydrogen bonds

Question 20

What is the maximum repeats and runs?

Answer 20

4 for both

Question 21

This avoids a di-nucleotide from occurring several times consecutively.

Answer 21

Repeats

Question 22

This avoids a long run of single bases.

Answer 22

Runs

Question 23

This type of amplification yields different bands of different sizes. How do you interpret such result?

Answer 23

Non-specific amplification; Poorly designed

Question 24

This type of amplification yields a single band. How do you interpret such result?

Answer 24

Specific Amplification; Ideal result

Question 25

What are the three types of secondary structures?

Answer 25

1. Hairpin
2. Self-dimer
3. Cross-dimer

Question 26

This type of secondary structure holds intramolecular interactions within the primer.

Answer 26

Hairpin

Question 27

This type of secondary structure holds intramolecular interactions between the same sense primers.

Answer 27

Self-dimer

Question 28

This type of secondary structure holds intramolecular interactions between the sense and antisense primers.

Answer 28

Cross-dimer

Question 29

This type of secondary structure is where forward and reverse primers bind to each other instead of the targeted primer region.

Answer 29

Cross-dimer