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

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)