Primer design for cDNA amplification

Examples given are suited to TaqMan primer design for mouse genes.

  1. Search Entrez for the nucleotide sequence of interest. In the Limits sections, select Only From: RefSeq.

  2. Choose the result from the appropriate organism (Mus musculus). Copy the RefSeq Accession Number (probably begins with NM_).

  3. Enter the RefSeq Accession Number into ELXR, and select the appropriate organism (Mouse). This aligns the cDNA against the genomic DNA sequence, and produces a graphical representation of the distribution of exons.

  4. ELXR also generates primers for amplifying specific exons, using the genomic sequence of the introns on either side of the desired exon. To amplify across exons takes a bit more work:

  5. At the bottom of the ELXR output, select "View mRNA/EST to genomic sequence alignment ". This shows the exon boundaries in the cDNA sequence.

  6. Copy the cDNA sequence from Entrez, and paste it into Primer3. Select an appropriate mispriming library (Rodent + Simple). In the Targets field, add numbers that represent one of the exon boundaries. For example, if EXLR showed exons at 1-154 and 155-237, you'd enter '153,4' as a target.

  7. Set an optimal product size (200bp), primer size (20bp), primer Tm (60ËšC), primer GC content (50%), and adjust the minimum and maximum values to be close to these optimum values.

  8. Press 'Pick Primers', and the best primer pair should be shown with a graphical representation of their placement on the cDNA sequence. A number of additional oligonucleotides are also suggested.

  9. If ordering custom primers from Invitrogen, sequences can be entered and ordered online.