Question

 How to Design Primers for PCR

The lab report should also have the following criteria Introduction, Principle, and procedure.

Answer 1

Introduction and principle:-

PCR is often called as one of the most beneficial discovery of 20th century. A very minute amount of genetic material can be amplified manifolds and then used for various applications. PCR mainly involves three steps:- denaturation, annealing and extension. Primarily, the DNA is denatured coverting dsDNA to single stranded then the primers are annealed to complementary regions of ssDNA and then they are extended by the action of DNA polymerase.

PROCEDURE:-

While running a PCR reaction, oligonucleotide primers are necesaary. They are synthesized chemically by joining nucleotides together. While designing a primer, size of the primer is very important. A short primer is used for the amplification of small fragments such as DNA fragments and long fragments if primers are used to amplify long fragments such as eukaryotic genome DNA sample. The length of primer should not be too short or too long because short primers give inaccurate and non specific results whereas long primers decrease the rate of amplification.

These following things should be kept in mind while designing a primer:-

1) length should be 18-24 bases

2) 40-60% of GC content

3) melting temperature of 56-60 degrees C.

4) primer pairs should not have complementary region.