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Tips for using BLAST to locate PCR primers

Need a quick way to find the location of primers within a gene or the expected size of the resultant PCR product? In this tip we show you how to get this information using BLAST.

NCBI’s BLAST is an incredibly powerful tool that efficiently queries the massive Genbank database. However, due to the heuristic nature of BLAST and removal of low complexity data, queries for short sequences like primers often return incomplete data. The following tips will improve these results:

  1. Concatenate the two primer sequences into one sequence separated by 5–10 Ns and enter into BLAST sequence box.

    Note: both primers can be in the same orientation as BLAST will search both strands for matches.
  2. Before submitting, narrow the search by selecting the species, if known; otherwise, choose Nucleotide Collection (nr/nt). If you’re looking for RT-PCR primers, select the reference mRNA sequences (refseq_mRNA) database.
  3. Under Program Selection, select the Somewhat similar sequences (blastn) program.
  4. Under Algorithm parameters, decrease word size to 7, increase expect threshold to 1000, and turn off the low complexity filter.

In the example below, the results with a line connecting the 2 boxes indicate the 2 primers are in the same sequence (Figure 1A). Clicking on these results shows location within the sequence (between bases 1005 and 1766 of the Mre11 transcript in yeast), and indicate the expected PCR product should be 762 bp in length (Figure 1B).

BLAST Results Show Primer Positioning and PCR Fragment Size

Figure 1. BLAST Results Show Primer Positioning (A) and PCR Fragment Size (B). (A) Only the best 3 alignments are shown (blue lines at the bottom). Selecting one of them provides the actual sequence alignment as shown in Panel B. Alignment of the 2 portions of sequence, showing 100% identity.

Additional reading

Design efficient PCR and qPCR primers and probes using online tools—Simplify planning of your qPCR experiments using IDT free, online tools for oligonucleotide analysis and PCR primer design. This article provides an overview of our predesigned qPCR assays and the basics of designing customized PCR primers and hydrolysis probes with the PrimerQuest® Tool.

Exon Numbering—Not As Easy As 1, 2, 3...Genes with multiple transcripts cause confusion—Exon numbering and location data can differ across various software tools, including with NCBI's gene database. For example, exons within alternatively spliced transcripts are sometimes individually numbered, with no consistent gene-based numbering system across these transcripts for identifying exons. Learn how IDT exon location information gives each exon a unique number and how that compares with the NCBI naming system.

qPCR Probes—Selecting the Best Reporter Dye and Quencher—Read these recommendations for choosing dyes and quenchers, taking into account instrument compatibility and multiplex probe applications.

Or just browse the qPCR article section of the DECODED Online newsletter.

Author: Adam Clore, PhD, is scientific applications scientist at IDT.

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