Create PCR primers optimized for length, tm, gc and free energy
Project description
primers
This is a tool for making PCR primers for DNA sequences. primers
' emphasis is on ease-of-use and DNA assembly. Adding additional sequence to 5' end of the FWD and REV primer, something common in creating fragments for Gibson assembly and Golden Gate cloning, is easy with primers
. Finally, it has a permissive MIT license where other primer design tools don't.
Installation
pip install primers
Usage
Python
from primers import primers
# add recognition sequences to FWD and REV primers
fwd, rev = primers("AATGAGACAATAGCACACACAGCTAGGTCAGCATACGAAA", add_fwd="GGTCTC" add_rev="GAAGAC")
print(fwd.fwd) # True
print(fwd.seq) # GGTCTCAATGAGACAATAGCACACACA; 5' to 3'
print(fwd.tm) # 62.4; melting temp
print(fwd.tm_total) # 68.6; melting temp with added seq (GGTCTC)
print(fwd.dg) # -1.86; minimum free energy of the secondary structure
# add from a range of sequence to the FWD primer
fwd, rev = primers("AATGAGACAATAGCACACACAGCTAGGTCAGCATACGAAA", add_fwd="GGATCGAGCTTGA", add_fwd_len=(5, 12))
print(fwd.seq) # AGCTTGAAATGAGACAATAGCACACACAGC
print(fwd.tm) # 62.2
print(fwd.tm_total) # 70.0
CLI
$ primers AATGAGACAATAGCACACACAGCTAGGTCAGCATACGAAA -f GGTCTC -r GAAGAC
dir tm ttm dg pen seq
FWD 62.4 68.6 -1.86 5.43 GGTCTCAATGAGACAATAGCACACACA
REV 62.8 67.4 0 4.8 GAAGACTTTCGTATGCTGACCTAG
$ primers --help
usage: primers [-h] [-f SEQ] [-fl INT INT] [-r SEQ] [-rl INT INT] [--version] SEQ
Create PCR primers for a DNA sequence.
Logs the FWD and REV primer with columns:
dir, tm, ttm, dg, pen, seq
Where:
dir = FWD or REV.
tm = Melting temperature of the annealing/binding part of the primer (Celsius).
ttm = The total melting temperature of the primer with added seq (Celsius).
dg = The minimum free energy of the primer (kcal/mol).
pen = The primer's penalty score. Lower is better.
seq = The sequence of the primer in the 5' to the 3' direction.
positional arguments:
SEQ DNA sequence
optional arguments:
-h, --help show this help message and exit
-f SEQ, --fwd SEQ additional sequence to add to FWD primer (5' to 3')
-fl INT INT, --flen INT INT
space separated min-max range for the length to add from 'add_fwd' (5' to 3')
-r SEQ, --rev SEQ additional sequence to add to REV primer (5' to 3')
-rl INT INT, --rlen INT INT
space separated min-max range for the length to add from 'add_rev' (5' to 3')
--version show program's version number and exit
Overview
Selecting primers for a DNA sequence is non-trivial because it's a multi-objective optimization problem. Ideally, pairs of primers for PCR amplification would have similar, ideal tms, low gc%s, low free energies (dgs) and lack off-target binding sites.
Scoring
In this module, the penalty for each possible primer, p, is calculated as:
PENALTY(p) =
abs(p.tm - opt_tm) * penalty_tm +
abs(p.gc - opt_gc) * penalty_gc +
abs(len(p) - opt_len) * penalty_len +
abs(p.tm - p.pair.tm) * penalty_tm_diff +
abs(p.dg) * penalty_dg +
p.offtargets * penalty_offtargets
Each of the optimal (opt_*
) and penalty (penalty_*
) parameters is adjustable through the primers.primers()
function. The primer pair with the lowest combined penalty score is chosen.
Given this module's emphasis on DNA assembly, additional sequences added to the FWD and/or REV primer are considered in the PENALTY calculation.
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