DNA Amplification Primers: Why Won't They Bind Effectively?
What is the reason that these two primers will not bind effectively to the region of DNA that we want to amplify?
a. The length of the two primers is between 20-30 base pairs long. The length is too short and not very specific.
b. The 3'-end of the reverse primer (3'-TTGGCCAATGG---5') is complementary to the forward primer (5'---AACCGGTTACC-3') and thus reduces the ability of the primers to bind to the targeted DNA sequence.
c. The 3'-end of the forward primer is complementary to the 5'-end of the reverse primer and thus reduces the ability of the primers to bind to the targeted DNA sequence.
d. The melting temperature range of the primers is similar to the DNA.
e. Guanine and Cytosine make up 60% of the total base pairs.
Answer: B
Explanation: The 3'-end of the reverse primer (3'-TTGGCCAATGG---5') is complementary to the forward primer (5'---AACCGGTTACC-3') and thus reduces the ability of the primers to bind to the targeted DNA sequence. There is possibility of hairpin loop formation or primer dimmers formation.
When designing primers for DNA amplification, it is crucial to consider the complementarity between the forward and reverse primers. In this case, the 3'-end of the reverse primer is complementary to the forward primer, which can lead to interference in the binding process.
The formation of hairpin loops or primer dimers can prevent the primers from effectively binding to the targeted DNA sequence. This interference can result in inefficient DNA amplification and affect the overall success of the PCR reaction.
Therefore, it is important to carefully design primers with non-complementary regions at the 3'-ends to ensure optimal binding and amplification of the DNA of interest.