Basic Applied Bioinformatics

CHAPTER 17Primer Designing – Basics

CS Mukhopadhyay and RK Choudhary

School of Animal Biotechnology, GADVASU, Ludhiana

17.1 INTRODUCTION

A primer is a short synthetic oligonucleotide, which is used to initiate amplification of DNA/RNA in a polymerase chain reaction (PCR). Literally, “to prime” means to “initiate” or “start”. In vivo, a short oligo‐sequence (i.e., the primer) is required, because the enzyme “DNA polymerase” has no capacity to initiate DNA replication without any primer. During this process of molecular photocopying, in vitro amplification of the target nucleotide sequence is initiated by a short complementary oligo.

Specificity and efficiency are two important factors for designing primers. Specificity of a primer pair is the ability of a PCR primer pair to amplify a specific product (i.e., no spurious amplification). The length and sequence of the oligo‐sequence pattern (repetitive or single copy, part of a multi‐gene family) of the template are factors that affect the specificity of primers. The efficiency of a primer pair refers to the fold increase of amplicon in each cycle, which should be ideally two folds in each cycle (practically, between 1.8 and 1.95).

17.2 OTHER IMPORTANT FEATURES FOR DESIGNING “GOOD” PRIMERS

17.2.1 Adding RE sites to primers

Restriction endonuclease (RE) site (4–6 nucleotides) is added at the 5′‐terminus of the oligo to use the amplicon in cloning and genetic engineering. Add 2–3 more bases before this RE site to facilitate the ...