mutate_spm: Calculate Primers for Point Mutagenesis

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

Description

The mutate function designs the necessary set of primers for the desired mutations. An example is given in the vignette at https://github.com/ipb-halle/GoldenMutagenesis/blob/master/vignettes/Point_Mutagenesis.md

Usage

mutate_spm(
  input_sequence,
  prefix = "TT",
  restriction_enzyme = "GGTCTC",
  suffix = "A",
  vector = c("AATG", "AAGC"),
  replacements,
  replacement_range = 2,
  binding_min_length = 4,
  binding_max_length = 9,
  target_temp = 60,
  cuf = "e_coli_316407.csv",
  fragment_min_size = 100
)

Arguments

input_sequence	The sequence which should be modified. This is an object of type character containing the sequence.
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
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: 2]
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]
cuf	The Codon Usage Table which is being used to select the codon for an exchanged amino acid. [default: e_coli_316407.csv]
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 Point Mutation vignette and design the primers
data(Point_Mutagenesis_BbsI_setup)
primers<-mutate_spm(input_sequence, prefix="TT", restriction_enzyme = recognition_site_bbsi, 
suffix = "AA", vector=c("CTCA", "CTCG"), replacements = mutations, binding_min_length=4 ,
binding_max_length=9, target_temp=60, cuf=cuf)

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