Running Primer3 to give Tm values only

Question

[yarrowmadrona]

I am trying to run primer 3 to analyze primers I have already designed. I want to see what Tm primer3 will give vs. other methods. I have an input file of 63 sequence ID in boulder I/O format. It only gives me calculated Tm for 25 of these. I really only want the calculated Tm for forward, reverse and probe. I have pasted a run that succeeded (first) and one that does not give Tm values (second).

INPUT:

SEQUENCE_ID=rs1045642
PRIMER_TASK=check_primers
SEQUENCE_PRIMER=CACACTTCCTGTTTGGGTTAGTTG
SEQUENCE_PRIMER_REVCOMP=GTCGAACACTTTCATCCCTTCCT
SEQUENCE_INTERNAL_OLIGO=CCTTTACCTGATCACCTGACCCTCC
PRIMER_PICK_LEFT_PRIMER=0
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=0
PRIMER_SALT_DIVALENT=3
PRIMER_SALT_MONOVALENT=50
PRIMER_DNTP_CONC=0.8
=

Output:

PRIMER PICKING RESULTS FOR rs1045642
    No mispriming library specified
    No internal oligo mishyb library specified
    Using 0-based sequence positions
    OLIGO            start  len      tm     gc%  any_th  3'_th hairpin seq
    LEFT PRIMER          0   24   61.88   45.83    0.00   0.00   36.79 CACACTTCCTGTTTGGGTTAGTTG
    RIGHT PRIMER       199   23   62.24   47.83    0.00   0.00    0.00 GTCGAACACTTTCATCCCTTCCT
    INTERNAL OLIGO      88   25   58.05   56.00    0.00   0.00    0.00 CCTTTACCTGATCACCTGACCCTCC
    SEQUENCE SIZE: 200
    INCLUDED REGION SIZE: 200

    PRODUCT SIZE: 200, PAIR ANY_TH COMPL: 0.00, PAIR 3'_TH COMPL: 0.00

        0 CACACTTCCTGTTTGGGTTAGTTGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
          >>>>>>>>>>>>>>>>>>>>>>>>                                    

       60 NNNNNNNNNNNNNNNNNNNNNNNNNNNNCCTTTACCTGATCACCTGACCCTCCNNNNNNN
                                      ^^^^^^^^^^^^^^^^^^^^^^^^^       

      120 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGG
                                                                   <<<

      180 AAGGGATGAAAGTGTTCGAC
          <<<<<<<<<<<<<<<<<<<<

    KEYS (in order of precedence):
    >>>>>> left primer
    <<<<<< right primer
    ^^^^^^ internal oligo

Here is an example that does not give me an Tm values: INPUT:

SEQUENCE_ID=rs12248560
PRIMER_TASK=check_primers
SEQUENCE_PRIMER=CCACGATGGGCATCAGAAGAC
SEQUENCE_PRIMER_REVCOMP=AGGGAGACCCTGGGAGAACAG
SEQUENCE_INTERNAL_OLIGO=TCAGCTCAAATCCCAGTTCTGCCA
PRIMER_PICK_LEFT_PRIMER=0
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=0
PRIMER_SALT_DIVALENT=3
PRIMER_SALT_MONOVALENT=50
PRIMER_DNTP_CONC=0.8
=

OUTPUT:

PRIMER PICKING RESULTS FOR rs12248560

No mispriming library specified
No internal oligo mishyb library specified
Using 0-based sequence positions
NO PRIMERS FOUND

SEQUENCE SIZE: 200
INCLUDED REGION SIZE: 200


    0 CCACGATGGGCATCAGAAGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
   60 NNNNNNNNNNNNNNNNNNNNNNNNNNNNTCAGCTCAAATCCCAGTTCTGCCANNNNNNNN
  120 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNC
  180 TGTTCTCCCAGGGTCTCCCT

Answer 1

[Charles Plessy]

Primer 3 provides additional command-line tools, among which oligotm, that you may find useful. (see this link to its manual page in Debian)

[yarrowmadrona]

Thank you, This is very helpful.

Answer 2

[h.mon]

Primer3 is a software to design primers given a number of constraints (primer size, primer GC%, min and max Tm, Tm difference between primers, and so on). What is happening is Primer3 is not happy with some of the primers you designed, as they probably fail on one or more of these constraints.

You could use some software designed only for Tm calculation, or you could relax Primer3 constraints to see if it "designs" your primers for you.

[boczniak767]

It's possible to use very permissive parameters so provided primers would be accepted and Tm will be shown. But there are few methods to compute it, so be aware of that. You should determine experimentally which Tm computation methods works best with your thermocycler.

[yarrowmadrona]

good point. As an aside I have kind of a strange situation in that the primers are designed by another software that takes into account proprietary chemical modifications included in the primers that are designed to raise the Tm. I can't input these modifications into primer3 and I can't access this proprietary software to run on the command line. I am trying to to compare Tm differences in this "blackbox" software and primer3 so that I can make my own adjustment when designing primers with primer3.

[yarrowmadrona]

Thanks, I was thinking of this for a couple of primer designs as Primer3 reports that they are too long. However, in many cases there is nothing printed that tells me what is wrong.