The Primer Generator

The Primer Generator is an automated generator of primers for site-directed mutagenesis. The program analyzes the original nucleotide sequence and desired amino acid sequence and designs a primer that either has a new restriction enzyme site or is missing an old one. This allows for faster sorting out of mutated and non-mutated sequences.

Read more about The Primer Generator in BioTechniques:
The Primer Generator: A Program that Facilitates the Selection of Oligonucleotides for Site-Directed Mutagenesis
BioTechniques 1999; 26:660-668

We recommend that you enter 7 base pairs on either side of the intended mutation so that The Primer Generator can take advantage of restriction enzymes with extra large sites or ones that cut away from the intended mutation site in a reasonable amount of time.

The Primer Generator is case-insensitive. All non-alphanumeric characters will be automatically eliminated from the input before processing (i.e. triplets can be separated with spaces, commas, etc.). Nucleotide sequences are translated according to the universal genetic code.

Please use one-letter code for amino acid sequences and "*" to indicate stop codon.

If you are new to this site, take a look at SAMPLE OUTPUT or at the outline of the program to see what you are in for.

If you use this program for your work, feel free to tell us about your experiences. Your feedback will help us make it better!

Warning:

This program can take up to several minutes to generate primers and produce hundreds of kB of output. This can be avoided by:

Submitting original sequence 15 or less bp (5 or less amino acid residues) long
Selecting the smallest possible maximum number of substitutions (see below)
Disallowing use of restriction enzymes with sites less than 5 base pairs
Limiting the repertoire of restriction endonucleases to the most frequently used enzymes

Use restriction enzymes with site size less than five base pairs?
(Is only applicable if Most Frequently Used Enzymes is not selected)

Limit to the most frequently used enzymes?

Maximum number of substitutions compared to original sequence:

Enter the original nucleotide sequence (7 base pairs on both sides of the indended mutation site recommended, unless only most frequently used enzymes are used) starting in the translated frame here:

Enter the desired amino acid sequence using the standard single-letter amino acid code (use * for stop codon) here:

Authors:

	Joseph Lawler
	Alexander Turchin