GetPrimers

A generalized PCR-based genetic targeting primer designer

Targeted genome

Choose a pre-built genome assembly & annotation:

Genome assembly

Annotation GFF style

NCBI

Ensembl

Or specify a customized genome assembly & annotation:

Upload assembly (*.fa, *.fasta, *.fa.gz, *.fasta.gz)

Upload GFF (*.gff3, *.gff3.gz)

Plasmid

Choose a pre-built plasmid cassette

Or upload a user-defined selection cassette sequence of the targeting plasmid (*.fa, *.fasta, *.fa.gz, *.fasta.gz)

Gene list:

All genes

Design primers for all genes

Strategy:

Targeting type:

Email address:

Product size and primer location

The number of candidate primers for the initial search
The minimal primer size bp.
The maximal primer size bp.
The minimal product size for gene targeting and verification PCR bp.
The maximal product size for gene targeting and verification PCR bp.
G1 is - bp upstream of the insertion site.
V1 is - bp upstream of the insertion site.
G2 is - bp downstream of the insertion site.
V2 is - bp downstream of the insertion site.
Size of the homologous region in P1 matching with the targeted genome (for the long primer strategy only) bp.
Size of the homologous region in P2 matching with the targeted genome (for the long primer strategy only) bp.
Size of primer common sequence upstream of target region, which is homologous to insertion cassette bp. Size of primer common sequence downstream of target region, which is homologous to insertion cassette bp.

Specificity

GetPrimers considers computated primers with ≥ total mismatches with off-target genomic regions as primers having on-target specificity.
For computated primers that have ≥ total mismatches with off-target genomic regions, if ≥ mismatches are within the last bp at the 3' end, GetPrimers also consider the corresponding primers having on-target specificity.

Dimer

GetPrimers considers pairs of primers that have > matches within the last bp at the 3' end to be dimer-prone.

The melting temperature (Tm) calculation

The concentration of primers (nM)
The concentration of monovalent cation (mM)
The concentration of divalent cation (mM)
The concentration of divalent cation (mM)

Other options

Perform repeat masking.
Run Blast against targeted genome for detecting non-specific primers.
De novo design of plasmid matching primers.
Thermodynamic model is used for oligo-oligo interactions and hairpins.

Presented by EvomicsLab@SYSUCC
yuejiaxing[at]gmail[dot]com