Primer Design Tutorial

Primers are short pieces of DNA used in a biological technique known as the polymerase chain reaction (PCR). PCR is used by molecular biologists to amplify gene sequences for more sophisticated scientific analysis, such as DNA sequencing, cloning or various biotechnological applications. Primer design is a skill that should be mastered by anyone wishing to study genes, and requires a solid understanding of biology and genetics. Although the rules for primer design are relatively straightforward, the complexities of genetics, experimental platforms and computing systems have resulted in hundreds of advanced software programs that can be used for primer design.

Instructions

  1. Primer Design Tutorial

    • 1

      Get your gene identity for Entrez Gene database. Search for the "REFSEQ" identity of your gene by simply entering the name of your gene into the search box. Obtain the Refseq cDNA identity of your chosen gene (e.g. "NM_136342").

    • 2

      Download the "FASTA" sequence. This is the nucleotide content of your gene represented as alphabets and is basically a text file. Look for the "greater than" symbol (>), which signals the beginning of the gene sequence. Either select, copy and paste the entire sequence or click on the "Download" button to retrieve it to your computer.

    • 3

      Obtain your gene sequence. Go to the BLAT or BLAST website (genome.ucsc.edu/cgi-bin/hgBlat) and paste your FASTA-format sequence into the search box. Click "Submit."

    • 4

      Use a professional primer design tool. These include software such as the freely-available perlprimer, or more sophisticated but commercial ones such as VectorNTI and MacVector. In perlprimer (perlprimer.sourceforge.net/), simply upload your sequence into the software and click "Find primers."

    • 5

      Check the primers. You should see forward and reverse primers ("primer pairs"), with numerical positions on the sequence, primer lengths, melting temperature and full primer sequence. Check that the primer pairs all the rules of primer design, such as weak primer-dimer formation, the presence of a GC clamp at the three-prime-end (3') of the primer, and that at least one primer is found spanning an intron-exon boundary.

    • 6

      Verify the products generated by the primers. Run an electronic PCR (e.g., NCBI's ePCR) to check the types or properties of gene products produced by the primers during a simulated PCR reaction.

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