SiteFind: A software tool for introducing a restriction site as a marker for successful site-directed mutagenesis

Paul M Evans,Chunming Liu

doi:10.1186/1471-2199-6-22

Paul M Evans, Chunming Liu

Open Access

https://doi.org/10.1186/1471-2199-6-22

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Abstract

BackgroundSite-directed mutagenesis is a widely-used technique for introducing mutations into a particular DNA sequence, often with the goal of creating a point mutation in the corresponding amino acid sequence but otherwise leaving the overall sequence undisturbed. However, this method provides no means for verifying its success other than sequencing the putative mutant construct: This can quickly become an expensive method for screening for successful mutations. An alternative to sequencing is to simultaneously introduce a restriction site near the point mutation in manner such that the restriction site has no effect on the translated amino acid sequence. Thus, the novel restriction site can be used as a marker for successful mutation which can be quickly and easily assessed. However, finding a restriction site that does not disturb the corresponding amino acid sequence is a time-consuming task even for experienced researchers. A fast and easy to use computer program is needed for this task.ResultsWe wrote a computer program, called SiteFind, to help us design a restriction site within the mutation primers without changing the peptide sequence. Because of the redundancy of genetic code, a given peptide can be encoded by many different DNA sequences. Since the list of possible restriction sites for a given DNA sequence is not always obvious, SiteFind automates this task. The number of possible sequences a computer program must search through increases exponentially as the sequence length increases. SiteFind uses a novel "moving window" algorithm to reduce the number of possible sequences to be searched to a manageable level. The user enters a nucleotide sequence, specifies what amino acid residues should be changed in the mutation, and SiteFind generates a list of possible restriction sites and what nucleotides must be changed to introduce that site. As a demonstration of its use, we successfully generated a single point mutation and a double point mutation in the wild-type sequence for Krüppel-like factor 4, an epithelium-specific transcription factor.ConclusionSiteFind is an intuitive, web-based program that enables the user to introduce a novel restriction site into the mutated nucleotide sequence for use as a marker of successful mutation. It is freely available from

Highlights

Site-directed mutagenesis is a widely-used technique for introducing mutations into a particular DNA sequence, often with the goal of creating a point mutation in the corresponding amino acid sequence but otherwise leaving the overall sequence undisturbed
After several rounds of PCR, the resulting mixture of newly-synthesized mutant constructs and template DNA is incubated with a methylation-specific endonuclease to remove the wild-type template DNA which contains methylated nucleotides
The user can use this information to design the appropriate primers for performing site-directed mutagenesis

Summary

Introduction

Site-directed mutagenesis is a widely-used technique for introducing mutations into a particular DNA sequence, often with the goal of creating a point mutation in the corresponding amino acid sequence but otherwise leaving the overall sequence undisturbed. This method provides no means for verifying its success other than sequencing the putative mutant construct: This can quickly become an expensive method for screening for successful mutations. The mixture is transformed into competent bacteria, plated on an antibiotic-containing medium, and grown overnight to in order to allow individual colonies to grow

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