Skip to main content

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 is easy with primers. This is a common requirement for Gibson assembly and Golden Gate cloning. Finally, it has a permissive MIT license where other primer design tools, like Primer3, don't.

Installation

pip install primers

Usage

Python

from primers import primers

# add enzyme recognition sequences to FWD and REV primers: BsaI, BpiI
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: [5, 12] bp
add_fwd = "GGATCGAGCTTGA"
fwd, rev = primers("AATGAGACAATAGCACACACAGCTAGGTCAGCATACGAAA", add_fwd=add_fwd, 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] [-t SEQ] [--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's secondary structure (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       additional sequence to add to FWD primer (5' to 3')
  -fl INT INT  space separated min-max range for the length to add from '-f' (5' to 3')
  -r SEQ       additional sequence to add to REV primer (5' to 3')
  -rl INT INT  space separated min-max range for the length to add from '-r' (5' to 3')
  -t SEQ       sequence to check for offtargets binding sites
  --version    show program's version number and exit

Overview

Creating primers for a DNA sequence is non-trivial because it's multi-objective optimization. Ideally pairs of primers for PCR amplification would have similar tms, GC ratios close to 0.5, high minimum free energies (dg), and a lack off-target binding sites. In primers, like Primer3, this is accomplished with a formula in which undesired primer characteristics are penalized. The primer pair with the lowest penalty score is created.

Scoring

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_offtarget

Each of the optimal (opt_*) and penalty (penalty_*) parameters is adjustable through the primers.primers() function. The defaults are below.

opt_tm: float = 62.0
opt_gc: float = 0.5
opt_len: int = 22
penalty_tm: float = 1.0
penalty_gc: float = 3.0
penalty_len: float = 1.0
penalty_tm_diff: float = 1.0
penalty_dg: float = 2.0
penalty_offtarget: float = 20.0

Offtargets

Offtargets are defined as a subsequence within one mismatch of the last 10bp of a primer's 3' end. This is experimentally supported by:

Wu, J. H., Hong, P. Y., & Liu, W. T. (2009). Quantitative effects of position and type of single mismatch on single base primer extension. Journal of microbiological methods, 77(3), 267-275

By default, primers are checked for offtargets within the seq parameter passed to primers.primers(seq). But the primers can be checked against another sequence if it's passed through the offtarget_check argument. This is useful when PCR'ing a subsequence of a larger DNA sequence; for example: a plasmid.

seq = "AATGAGACAATAGCACACACAGCTAGGTCAGCATACGAAA"
parent = "ggaattacgtAATGAGACAATAGCACACACAGCTAGGTCAGCATACGAAAggaccagttacagga"

# primers are checked for offtargets in `parent`
fwd, rev = primers(seq, offtarget_check=parent)

Project details


Download files

Download the file for your platform. If you're not sure which to choose, learn more about installing packages.

Source Distribution

primers-0.2.1.tar.gz (12.2 kB view hashes)

Uploaded Source

Built Distribution

primers-0.2.1-py3-none-any.whl (13.1 kB view hashes)

Uploaded Python 3

Supported by

AWS AWS Cloud computing and Security Sponsor Datadog Datadog Monitoring Fastly Fastly CDN Google Google Download Analytics Microsoft Microsoft PSF Sponsor Pingdom Pingdom Monitoring Sentry Sentry Error logging StatusPage StatusPage Status page