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primer3-org / primer3

Primer3 is a command line tool to select primers for polymerase chain reaction (PCR).
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how to determine the integer input for SEQUENCE_OVERLAP_JUNCTION_LIST #53

Closed juechenyang closed 2 years ago

juechenyang commented 2 years ago

Hi,

I have a question regarding to how to determine the integer input for SEQUENCE_OVERLAP_JUNCTION_LIST. Suppose I have a gene which have 3 exons with their length of 10,12,15 respectively. In this case, what should be the input integer for SEQUENCE_OVERLAP_JUNCTION_LIST? Is it something like "10 22 37" where 22 is first two exon's length and 37 is the all three's. If yes, what if this gene is on reverse strand? Is this going to be a different integer list?

Thank you

untergasser commented 2 years ago

See the Manual: https://primer3.org/manual.html#SEQUENCE_OVERLAP_JUNCTION_LIST

The idea is to use a cDNA without introns, so they are removed from the sequence. "10 22 37" means 10-11, 22-23 and 37-38 are the junctions where the introns were before removal.

Best,

Andreas

juechenyang commented 2 years ago

Thanks! So these numbers are indicating positions on DNA or cDNA? For example, I have a gene which has three exons that are sequentially distributed on genome at 10000-10010, 20000-20020, 30000-30060. Which of the following inputs are correct for SEQUENCE_OVERLAP_JUNCTION_LIST

  1. 10010 20020, 30060
  2. 10 30 100
  3. 10 20 60

Thanks so much for your time!

untergasser commented 2 years ago

Primer3 takes one sequence. It does not know about introns and exons. The positions are for this sequence and it tries to overlap them as defined by the other parameters.

So for your example you remove the introns from the sequence and use: 10000 20000 30000

Best,

Andreas