Deborah Dawson ([email protected]) Sheffield Molecular Genetics Facility 18th November 2003 WHEN MIICROSATELLITE PRIMERS FAIL – WHAT TO CHECK – BACK TO BASICS 1. Linker Contamination If an enriched library was prepared – Perform a NCBI nBLAST check to search for linker contamination. Ensure EMBL sequences and the sequence from which the primers were designed have had the linker removed. Check where primers complement on the full sequence. Edit EMBL records if needed – Redesign primers if they overlapped with linker. 2. Vector Contamination BLAST check for vector contamination. Ensure EMBL sequences and the sequence from which the primers were designed are vector free. Check where primers complement on the full sequence. Edit EMBLrecords if needed. Redesign primers if they overlapped with vector. 3. Multiple Inserts (Obvious ones) Check for double inserts. Search for GATC (if MboI) using “Find” in WORD or using GENEJOCKEY in sequence editor files and treat each separate insert as a separate microsatelite. 4. Blunted-ended Multiple Inserts (Hidden ones) Check for a lack of the enzyme restriction site (GATC) in seq. If missing at very front or/and very end of sequence this suggests there are blunt ended fragments. If present, this suggests multiple inserts which are undetected and probably leading to primer failure. 5. Repetitive sequences Multiple copies of same or similar sequences (often only on one flank of sequence) will result in the primers failing. BLAST all sequences (which must be in EMBL and released to the public) to check for within library duplication and see if “families” of sequence types are present. 6. Primer Sequence Identity BLAST the primer sequences to check the primers designed actually “fit” on the sequence from which they were supposedly designed. 7. Poor Primer Sequence Design Check using PRIMER3 (new version on web) if good primers were design – paste in full seq and the forward and reverse primers and see if PRIMER3 approves of them. Check using PRIMER3 if the size of product was accurately recorded and if the annealing temperatures were recorded properly. 8. Species identity / contamination BLAST to check the microsatellite are from the correct species and not been isolated from human or other species contamination. Eg A bird species with normally pick up some degree (even if weak) of sequence homology to other birds in 1 of say 10 of sequences BLAST checked. Mammals are easy to check and will have lots of matches to other mammals of same family (lot a of dog, cat, mouse and human sequences are on the database).