What is the difference between the forward and reverse primer?
The Correct Answer and Explanation is:
Correct Answer:
The forward primer binds to the 3’ end of the antisense (template) strand and has the same sequence as the sense strand. The reverse primer binds to the 3’ end of the sense strand and has a sequence complementary to the sense strand.
Explanation:
Primers are short sequences of nucleotides used in polymerase chain reaction (PCR) to initiate DNA synthesis. Two primers are needed for a successful PCR: the forward primer and the reverse primer. Although they serve a similar purpose, their orientations and target strands are different.
The forward primer is designed to bind to the 3’ end of the antisense strand, which is also known as the template strand. Since DNA synthesis occurs in the 5’ to 3’ direction, the forward primer binds to the 3’ end of this strand and allows the DNA polymerase to synthesize a new strand in the 5’ to 3’ direction. The sequence of the forward primer is identical to a portion of the sense strand because it binds to the complementary antisense strand.
In contrast, the reverse primer binds to the 3’ end of the sense strand, which is the non-template strand. Because the reverse primer must also allow synthesis in the 5’ to 3’ direction, it is complementary to the sense strand. This primer binds in the opposite direction compared to the forward primer and allows polymerase to synthesize a strand complementary to the sense strand.
Together, these primers bracket the DNA region of interest, enabling amplification of a specific segment. Designing primers correctly is crucial for the specificity and efficiency of PCR. If either primer is incorrectly designed, the reaction may fail or result in non-specific amplification. The orientation, sequence, and melting temperature of both primers must be considered to ensure they bind accurately and initiate proper replication.
