[08] Primer Design Flashcards
What is a primer in the context of molecular biology?
A short strand of RNA or DNA that serves as a starting point for DNA synthesis.
What is the purpose of a primer in PCR?
It binds to the DNA template and defines the starting point for DNA synthesis.
What are the key aspects to consider in primer design?
- Length of the primer
- Melting temperature (Tm)
- Specificity
- GC content
- Avoidance of self-complementarity
How does the length of the primer affect PCR?
A longer primer provides more specificity, but may also lead to more mismatches.
What is the importance of melting temperature (Tm) in primer design?
It determines the temperature at which the primer binds to the DNA template during PCR.
Define “Melting Temperature” (Tm).
The temperature at which half of the DNA duplex will dissociate to become single-stranded.
Why should primers be specific?
To ensure that they bind to the correct region of the DNA template.
Why is GC content important in primer design?
A higher GC content increases the melting temperature of the primer, making it more stable.
What does it mean if a primer is self-complementary?
The primer can bind to itself, leading to primer-dimer formation and reduced efficiency of PCR.
What is a primer-dimer?
A byproduct formed when primers bind to each other instead of the target DNA during PCR.
Define “Primer-Dimer”.
An artifact formed in the process of PCR by self-amplification of primers due to complementary sequences.
What is the role of a forward primer in PCR?
It initiates the first strand of synthesis in the 5’ to 3’ direction.
What is the role of a reverse primer in PCR?
It initiates the synthesis of the second strand, also in the 5’ to 3’ direction, but on the opposite DNA strand.
How do you determine the direction of a primer?
The direction is determined by the orientation of the 5’ and 3’ ends of the primer.
What is the consequence of mismatches in primer design?
Mismatches can prevent the primer from binding correctly, reducing the efficiency of PCR.
