Discussion
Amplification of a DNA fragment by PLR requires primers. Which of the following statements are correct for primers ?
(i) Primers with hairpin structure formation are not desirable
(ii) Primer sequence should not have long runs (\(\gt\)3) of a single nucleotide
(iii) GC content of the primer should be less than 20%
(iv) Both the forward and reverse primers must have high percentage of base complementarities
Answer: C
Statements (i) and (ii) are correct, so the answer is (i) and (ii).
For PCR primers (the paper prints 'PLR'), hairpins are avoided because a primer folded on itself cannot anneal to the template (i). Long runs of one base are avoided because they encourage mispriming and slippage (ii).
Statement (iii) is wrong: the recommended GC content is about 40 to 60%, giving a suitable melting temperature, and a GC content below 20% would make the primer bind too weakly. Statement (iv) is wrong as well: forward and reverse primers should not be complementary to each other, or they anneal together and form primer-dimers.
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