Software Features#

Parameter Setting#

The user parameter setting is possible for the inputs in PrimerDriver. The tool designs the desired mutagenic primer pairs according to user input and performs site-directed mutagenesis from single- or multiple-bases within the sequence.

When running primerdriver as standalone CLI, the user needs to modify the primerdriver/settings.json file to define custom values for the required parameters. The parameter defaults are as follows

{
    "Tm_range_min": 75,
    "Tm_range_max": 85,
    "gc_range_min": 40,
    "gc_range_max": 60,
    "length_min": 25,
    "length_max": 45,
    "flank5_range_min": 11,
    "flank5_range_max": 21,
    "flank3_range_min": 11,
    "flank3_range_max": 21,
    "forward_overlap5": 9,
    "forward_overlap3": 9,
    "terminate_gc" : true,
    "center_mutation": false,
    "primer_mode": "overlapping",
    "expression_system": "Homo sapiens"
}

When running primerdriver as part of a larger Python script, you can instead import the settings via

from primerdriver.config import get_settings

settings = get_settings()

then access/modify the individual parameters using dot notation, e.g.

settings.terminate_gc = False
settings.length_min = 20
settings.length_max = 55

then pass the updated settings when initializing the module

from primerdriver.primer_design import PrimerDesign

pd = PrimerDesign(*args, settings=settings)

The following definitions and considerations should be made for designing mutagenic primers:

  • Tm_range

    This is where the user sets the minimum and maximum allowable melting temperature (\(T_m\)) of primers in a PCR reaction. Depending on the type of mutation, the melting temperature is calculated differently. Estimation of the Tm of primers for base substitution is defined by

    \[T_m = 81.5 + 0.41(\%\textrm{GC}) - \frac{675}{N} - \%\textrm{mismatch}\]

    where \(N\) is the primer length in base pairs, and \(\%\textrm{GC}, \%\textrm{mismatch}\) are whole numbers. A modified formula is used for calculating \(T_m\) that intends to introduce deletions or insertions:

    \[T_m = 81.5 + 0.41(\%\textrm{GC}) - \frac{675}{N}\]
  • gc_range

    The GC content is calculated as

    \[\%\textrm{GC} = \frac{\textrm{# of G + # of C}}{\textrm{total length of primer}}\]

    and the range of GC in a primer is to be set by the user. The user can follow the percentage as prescribed by a protocol or the user can just set the range from 0 to 100.

  • length

    The total length of the desired mutagenic primer

  • flank5_range

    This is the length of the region that is to the left of the mutation when read in standard sequence notation.

  • flank3_range

    This refers to the length of the region that is to the right of the mutation when read in 5’ to 3’.

  • forward_overlap5

    When generating overlapping primers, this is where the user sets the minimum number of overlapping bases of the 5’ end of the forward primer to the reverse primer.

  • forward_overlap3

    This sets the minimum number of base overlaps from the 3’ end of the forward primer to the reverse primer.

  • terminate_gc

    Set in boolean values, this refers to having a GC clamp at both ends of the primers.

  • center_mutation

    This sets the mutation is centered within the primer. Flanking regions differ by at most one base pair. These are set by boolean values.

  • primer_mode

    This gives the user the choice of having overlapping primers or complementary primers. Both modes use protocol from QuikChange™ Site-Directed Mutagenesis Kit by Stratagene®.

    • Complementary

      The values for the flanking regions will just be applied to the forward primer. The reverse primers will just be the reverse complements of the forward primers.

    • Overlapping

      Both the forward and reverse primers will be screened for the flanking region values. Forward overlap values will also be applied when generating the primer pairs.

  • expression_system

    When using Protein based primer design, the user can choose from a number of commonly used model organisms used in molecular and cell biology. This enables the tool to generate primers that use the optimum codon for the chosen organism.

Input format#

In PrimerDriver, sequences can be added either by copying/pasting into the input cursor. The tool also accepts one or multiple sequences when uploaded in FASTA format. The input sequence must match the DNA sequence that will serve as template for PCR. Before processing the sequences, a basic input emending (FASTA headers, unsupported characters) is performed.

Choosing an interface#

As a powerful and useful feature in DNA engineering, the Web server offers the possibility of designing primers for site-directed mutagenesis. The tool can accommodate both DNA and protein sequences to incorporate base pair insertions, deletions, and substitutions as specified by the user. This can cater to an array of primer designs for site-directed single mutagenesis. PrimerDriver also lets you choose from two different command-line modes:

  • Interactive mode

    Guides the user through a step-by-step input prompt. Primarily aimed towards demonstration purposes and first-time users of the program.

  • Single-command mode

    Requires the user to include all the inputs and arguments needed to perform the task in a single command. Primarily aimed towards more advanced tasks such as batch primer design.

Choosing primer design modes#

PrimerDriver asks you to choose from three different modes when using the command-line interface:

  • Primer characterization

  • DNA-based primer design

  • Protein-based primer design