I have a full published plant genome (280Mb). I ran MISA to identify microsatellite regions and found approx 100,000 hits. I then formatted the output files for Primer3 and ran with default settings. From this I got zero suggested primers (I increased maximum primer size and decreased minimum melting temperature as suggested - still no hits). Thus, I set PRIMER_EXPLAIN_FLAG=1 and have 200000+ (forward & reverse) files. As I understand all these discarded "primers" listed in these files failed. Could someone explain why they failed or why a complete genome would yield no hits. For example, for the following discarded primer has the following metrics:
21 20 0 50.00 59.490 3.00 1.00 0.510
Quality is 0.51 (as I understand this is good - i.e. low) - from the example file with the software the selected/outputted primers have quality of 3 (which is worse as I understand); self-annealing values are also low/good (3 & 1); GC is 50% & temp is 59.49 - are these the reasons it is not accepted under defaults?
How should I select the best potential primers from these files?
Any help much appreciated thanks
Clive
Aren't microsatellites repettitive regions and therefore sequences are highly similar? Do surprise you get no unique primers I would say. Is this for qPCR?
Not sure about PCR method, I was asked to find some msat primers from a published genome - identifying microsats was easy, just don't understand the output of Primer3 and find its docs pretty unclear.