From Kyle Harris and Alison Nairn: 4/12/07

Protocol for Primer Design for “Glycotranscriptome” analysis

Steps for primer design:

1.  Copy/Paste Accession Number from gene list into PubMed Nucleotide search.

2.  Copy/Paste the full GenBank record from file into new Word document and save as the GeneList # designation for the given gene. Separately copy/paste the full GenBank record into a HTML file and name the file “a.htm” for incorporation into the “Glycotranscriptome” web site.

3.  Click on CDS link and copy/paste coding sequence into the Word file. Similarly, copy/paste the coding sequence record into a HTML file and name the file “b.htm” for incorporation into the “Glycotranscriptome” web site.

4.  Copy/Paste coding sequence into BLAST search window (select “REFERENCE only”, select genome to search).

5.  Copy/Paste the resulting data from the web browser into the Word gene file. Copy/paste the genome organization record into a HTML file and name the file “c.htm” for incorporation into the “Glycotranscriptome” web site.

6.  Select exon sequence to use for primer design (usually the largest coding exon).

7.  Copy/ Paste exon sequence from coding sequence into new subheading “Exon used for primer design” in the Word gene file and also paste into a new HTML file named “d.htm”.

8.  Two methods have been used for Primer Design: Molecular Beacon software or Primer3 software. We generally use Primer3 for our primer design, but some of the early primers were designed and validated using the Molecular Beacon software (i.e. some of the glycosyltransferase genes).

9.  Primer 3 approach: Open Primer 3 website and paste the exon sequence into “source sequence box” and change default settings as described:

  1. Restrict primer melting temperature (Tm) to 59-61ºC.
  2. Restrict primer length to 19-21 base pairs.
  3. Change the following parameters:
  4. Self Dimer Max Delta G (3’end) : 5 kcal/mol
  5. Self Dimer Max Delta G (Internal) : 6 kcal/mol
  6. Run/Repeat (dinucl.) Max Length : 3 bp
  7. G/C clamp-Target consec. G/C’s at 3’ end : 1
  8. Amplicon Length : 65-75 base pairs
  9. Max Primer Pair Tm Mismatch :1°C
  10. %GC Content : 20-80%
  11. Number to return : 5 (can be changed as needed)

10. Copy/Paste Primer data into the Word gene file and to the “d.htm” file.

11. Copy/paste the candidate primer sequences to the bottom of the Word gene file and BLAST search each against the specific genome using “exact nucleotide match”.

12. Check highly similar matches to ensure the primers don’t match other genes.

Molecular Beacon Primer Design Protocol

1.  Enter exon sequence.

2.  Restrict primer temperature to 59-61ºC.

3.  Restrict primer length to 19-21 base pairs.

4.  Change the following parameters:

  1. Hairpin Max Delta G (3’end) - 2 kcal/mol
  2. Hairpin Max Delta G (Internal) - 3 kcal/mol
  3. 3’ end max delta G - 10 kcal/mol
  4. Self Dimer Max Delta G (3’end) - 5 kcal/mol
  5. Self Dimer Max Delta G (Internal) -6 kcal/mol
  6. Run/Repeat (dinucl.) Max Length - 3 bp
  7. G/C clamp-Target consec. G/C’s at 3’ end – 1
  8. Amplicon Length – 65-75 base pairs
  9. Max Ambiguous Bases in Amplicon – 0
  10. Max Primer Pair Tm Mismatch – 1C
  11. Cross Dimer Max Delta G (3’ end) – 5 kcal/mol
  12. Cross Dimer Max Delta G (Internal) – 6 kcal/mol
  13. %GC Content – N/A