Mutagenic Primer Design

Introduction

This tool is designed to generate primers for quick-change missense mutations easily on google Colab. Below are the instructions for preparing the necessary input files.

Preparing Input Files

DNA Sequence File (FASTA Format)

• Example Filename: Shaker_FL.fa
• Content: Start with a header line beginning with >, followed by your sequence description. Write the DNA sequence in standard nucleotide codes (A, C, G, T) on the following lines. Sequence must be in frame.
• Note: If the first codon does not correspond with the first aminoacid, this can be indicated by modifying the starting_residue value

Mutations File (CSV Format)

• Example Filename: mutations.csv.
• Content: List mutations as comma-separated values. Each line should be in the format [position],[amino acid], where [position] is the residue number and [amino acid] is the desired amino acid using the single-letter code. Other options migth include "*" for ochre stop codon, "-" for amber and "X" for random mutation.

Codon Table File (CSV Format)

• Example Filename: codontableXlaevis.csv.
• Content: Include a mapping of amino acids to their respective codons. The first column lists the amino acids (single-letter code), followed by columns listing the high frequency codons.

Running the Notebook

Execute the cells in the notebook in order, ensuring that each cell completes successfully before proceeding to the next.

Output

• Filename:primers.csv
• Content: List of primers as comma-separated values. First column list the name of the primer (position_aa_Fw), second column is the primer sequence, the third column is the calculated Tm.