CHAPTER 19Quality Checking of the Designed Primers

CS Mukhopadhyay and RK Choudhary

School of Animal Biotechnology, GADVASU, Ludhiana


Biocomputationally designed primers may produce secondary structures such as homo‐ and hetero‐dimers and hairpin loop structures. These structures interfere with the efficiency and specificity of the primers, and produce noise in SYBR green chemistry‐based real‐time PCR assays. The online OligoAnalyzer Version 3.1 tool (Integrated DNA Technology: can be used to detect possible secondary structures produced by primer(s).


To determine the quality of the designed primers for specificity to the template and possibility of secondary structure formation.


Open the IDT Oligo Analyzer Version 3.1 online tool using the URL:

19.3.1 Sequence box

This is located at the left top of the page. Each of the primer sequences (i.e., Forward and Reverse primers in 5′ to 3′ direction) is provided one at a time in this box (Figure 19.1). The software can understand a wide array of modified bases, including “standard bases” (symbols of the bases are not case‐sensitive), “mixed bases” (i.e., degenerate or wobble bases, in uppercase only), RNA (e.g., rA), and so on.

19.3.2 Parameters

The software uses the following parameters:

  • Target Type: either “DNA” or “RNA”. Select “DNA” for standard oligos.
  • Enter oligo concentration: Primer ...

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