PCR primer design using NCBI Primer-BLAST

Summary

The tutorial explains how to design PCR primers with NCBI Primer-BLAST. It covers basic primer-design principles and demonstrates a workflow using a messenger RNA accession number.

Primer-design guidelines

Primers should:

• Be 18–30 base pairs long. Shorter primers may be less specific, while longer ones may have excessively high melting temperatures.
• Have a GC content of 40–60%.
• Have forward and reverse primer melting temperatures (Tm) close to one another, with about 60°C as a general target.
• Avoid highly repetitive sequences.
• Avoid self-complementarity, especially at the 3′ end, to reduce primer-dimer formation.
• Avoid complementarity between the forward and reverse primers.
• Avoid mismatches with the template and runs of three or more Gs or Cs at the 3′ end.
• Be checked for specificity against an appropriate sequence database.

Primer-BLAST workflow

1. Enter the PCR template. Paste the target sequence into the template box or enter its accession number. The example uses an accession number for a messenger RNA target.
2. Set primer and product parameters. Choose a PCR product-size range appropriate for the experiment. For the real-time PCR example, the presenter sets the range to 70–200 bp. Larger product sizes may be appropriate for other applications, such as conventional PCR.
3. Choose exon-spanning design when appropriate. For gene-expression analysis, select the option requiring a primer to span an exon–exon junction. This can help avoid amplification from contaminating genomic DNA. The presenter notes that this setting is not relevant to every primer-design task.
4. Set specificity-checking parameters. Use an appropriate database; the example retains the RefSeq messenger RNA database setting. Select the correct organism—in this example, Homo sapiens.
5. Review and choose a primer pair. Primer-BLAST displays candidate pairs and information such as primer sequences, lengths, positions, Tm, GC content, and self-complementarity. Compare candidates and favor those with low self-complementarity, particularly at the 3′ ends. The example selects a pair with self-complementarity values of 4 and 3′-end complementarity values of 0 and 1.
6. Check the predicted target. Primer-BLAST compares suggested primers against the selected reference database. In the example, the predicted product is 132 bp, within the chosen range, and the alignment display indicates no mismatches at the positions shown.

Speakers and sources

• Speaker: Tutorial presenter from the Bio_Code channel
• Tool: NCBI Primer-BLAST
• Database: RefSeq messenger RNA