Basic Applied Bioinformatics

CHAPTER 18Designing PCR Primers Using the Primer3 Online Tool

CS Mukhopadhyay and RK Choudhary

School of Animal Biotechnology, GADVASU, Ludhiana

18.1 INTRODUCTION

The previous chapter (Chapter 17) discussed in brief the salient features of PCR‐primers. Here we will learn how to use Primer3, an online software tool, for designing PCR primers.

18.2 OBJECTIVE

To design primers for bubaline Dicer I cds, using the online primer designing tool Primer3.

18.3 PROCEDURE

18.3.1 Downloading a nucleotide sequence

Type the URL (www.ncbi.nlm.nih.gov/) to open the NCBI home page. Search the required nucleotide sequence, select the target sequence from NCBI nucleotide and save it in FASTA format in a text file.

18.3.2 Open Primer3 online tool

Open the Primer3 (version 4) software by using the URL http://primer3.ut.ee/.

18.3.3 Obtaining nucleotide sequence of interest

Paste the nucleotide sequence (in FASTA format) in the box for source sequence in the Primer3 page.

FIGURE 18.1 Setting the parameters of the Primer3 online tool for primer designing.

18.3.4 Set the required parameters

All the parameters mentioned on the Primer3 page have been explained, along with the optimal values, in Table 18.1.

TABLE 18.1 Primer3 parameters, description and their optimal values/options (http://ls23l.lscore.ucla.edu/Primer3/primer3web_help.htm).

SN	Parameter	Description	Optimal value