mutate_msd: Calculate Primers for Multiple Site Saturation Mutagenesis

In ipb-halle/GoldenMutagenesis: A tool to calculate and validate primers for Golden Gate Cloning

Description

The mutate_msd function designs the necessary set of primers for the desired mutations. Note that you can also select TGG in saturation mutagenesis to apply the 22c trick.

Usage

mutate_msd(
  input_sequence,
  codon = "NDT",
  prefix = "TT",
  restriction_enzyme = "GGTCTC",
  suffix = "A",
  vector = c("AATG", "AAGC"),
  replacements,
  replacement_range = 3,
  binding_min_length = 4,
  binding_max_length = 9,
  target_temp = 60,
  fragment_min_size = 100
)

Arguments

input_sequence	The sequence which should be modified. This is an object of type character containing the sequence.
codon	The desired type of MSD mutation [default: NDT]
prefix	Additional nucleobases in 5' position of the recognition site [default: TT]
restriction_enzyme	Recognition site sequence of the respective restriction enzyme [default: GGTCTC]
suffix	Spacer nucleotides matching the cleavage pattern of the enzyme [default: A]
vector	Four basepair overhangs complementary to the created overhangs in the acceptor vector [default: c("AATG", "AAGC")]
replacements	The desired substitutions. Can be a numeric vector or a list of character vectors with position and codon. See vignette("MSD3", package = "goldenmutagenesis") for examples.
replacement_range	Maximum distance in amino acid residues between two randomization sites to be incoporated into a single primer (reverse, end of the fragment) - has a cascading effect for following mutations [default: 3]
binding_min_length	The minimal threshold value of the length of the template binding sequence in amino acid residues [default: 4]
binding_max_length	Maximal length of the binding sequence in amino acid residues [default: 9]
target_temp	Melting temperature of the binding sequence in print('\u00B0')C [default: 60]
fragment_min_size	Minimal size of a generated gene fragment in base pairs [default 100]

Value

An object of class Primerset with the designed Primers.

Examples

#Load the setup of the MSD vignette and design the primers
data(MSD_BsaI_setup_lv2)
print(mutations)
print(recognition_site_bsai)
primers<-mutate_msd(input_sequence, prefix="TT" ,
restriction_enzyme=recognition_site_bsai, suffix="A", 
vector=c("AATG", "AAGC"), replacements=mutations, replacement_range=5,
binding_min_length=4 , binding_max_length=9, target_temp=60,
fragment_min_size=60 )

ipb-halle/GoldenMutagenesis documentation built on Sept. 1, 2020, 3:24 p.m.