PCR (Polymerase chain reaction)
See also How to design Primers for PCR and the YouTube tutorial.
This command is only valid for DNA sequences. GeneWarrior uses the Primer3 software (see references below) for Primer design and Tm (melting
temperature) calculation.
Primer design
Select a single DNA sequence as PCR template and click on "Primer design".
Set Rules
Define a PCR target by selecting the sequence using your mouse and clicking on "Set Target".
Primers will be created to flank the target. However, they cannot overlap it.
No Primers and Probes will be created on regions which are marked as Exluded. It makes sense to
exclude regions which have low sequence quality (e.g. the beginning of a Sanger sequence) or contain
repetitive elements.
Regions are excluded by selecting the sequence using your mouse and clicking on "Set as Exclusion".
Multiple regions can be excluded.
By using the "Force Primer to start at Position"-option, the 5′-end of the respective primer is
forced to start at the specified position. This of course limits the software to only choose the length of
the primer to satisfy your further constraints such as GC-content and melting temperature.
If both Left and Right primer positions are forced to a location, no target is necessary.
Set Parameters
This section allows you to set the parameters for the primer design and primer properties.
Product size: The minimum and maximum length of your PCR product (this includes the primers on both ends).
The bigger the range, the more options the software has to choose from possible primers. [This parameter corresponds to PRIMER_PRODUCT_SIZE_RANGE in Primer3]
Primer size: The minimum, optimum and maximum length of the chosen primers. The maximum is 35bp. [These parameters correspond to PRIMER_MAX_SIZE etc. in Primer3]
Primer melting temperature: The minimum, optimum and maximum melting temperature (Tm) in °C. The melting temperatures are computed according to SantaLucia, 1998 (see below) [These parameters correspond to PRIMER_MAX_TM etc. in Primer3]
Primer GC content: The minimum, optimum and maximum GC content of the chosen primers (in %). [These parameters correspond to PRIMER_MAX_GC_PERCENT etc. in Primer3]
Salt concentration: The concentration of monovalent salt cations in the PCR (usually KCl) in millimolar (mM). [This parameter corresponds to PRIMER_SALT_MONOVALENT in Primer3]
DNA concentration: The concentration of annealing oligos in the course of the PCR (in nanomolar (nM). [This parameter corresponds to PRIMER_DNA_CONC in Primer3]
Hybridization Probe
This option creates an additional oligo probe which binds on the PCR product itself. This is used
for qPCR/Real-time PCR.
The options are the same as above, but apply only to the probe reaction.
Choose Primers
If you selected a Target and chose your proper parameters, click on "Calculate Primers".
The software will create up to five primer pair suggestions to choose from. If your parameters are too stringent
to allow for primer design, an error message will detail on which step the software failed.
If everything is okay, you can inspect the primer pair suggestions using the drop-down box. The primers are
drawn into the sequence and the primer details such as melting temperature, positions etc. are displayed.
To select a chosen primer pair, click on "Export this Pair". The window will close and the PCR details
are imported into your project.
To check your chosen primer pair for unintended products, click on "NCBI PrimerBLAST". This opens NCBI
PrimerBLAST using your template and primer pair. Use the Primer Pair Specifity Checking Parameters to check for
possible mismatches of your primer pair to the genome or transcriptome of your species.
Reference
SantaLucia JR (1998) "A unified view of polymer, dumbbell and oligonucleotide DNA nearest-neighbor thermodynamics", Proc Natl Acad Sci 95:1460-65
Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386 Download software
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