Question

Following are the two statements regarding PCR primers.

Statement1: PCR primers must not have self-complementary regions.
Statement 2: Self-complementary regions result in hairpin structures, adversely affecting the PCR.

• A. Both the statements are true
• B. Statement 1 is true and statement 2 is false
• C. Statement 1 is false and statement 2 is true
• D. Both the statements are false

Answer 1

The correct option is A Both the statements are true

Polymerisation requires primers, be it in vitro or in vivo.
Primer is an oligonucleotide sequence that provides free 3’OH for starting DNA synthesis. Primers are very specific, bind to the specific DNA sequence it is made for and help to amplify the desired DNA stretch.
PCR primers are short pieces of single-stranded DNA usually around 20 nucleotides in length. For amplifying the desired dsDNA, two primers are used in each PCR reaction, both of the oligonucleotide sequences bind to both the strands of DNA just at the edges of the region which needs to be copied. The primers bind to the template strand by complementary base pairing.

Designing primers for a new sequence is the toughest of all in performing PCR reactions. To design a primer there are many things that need to be considered-

• The primer should have less guanine-cytosine base content at its 3’ end.
• The length of the primers must not be very long and also not very short, around 18-30 nucleotide bases.
• The melting temperature of the primers should be $65-70°C$.
• Complementary sequences in the primers should be avoided to prevent a chance of forming hydrogen bonds between them and forming secondary hairpin-like structures, which will eventually not bind to the template DNA and the process of chain reaction will get affected.