SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites

Abhijit Karnik, Rucha Karnik, Christopher Grefen*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

23 Citations (Scopus)

Abstract

Background: Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing.

Results: We have developed a program - 'SDM-Assist' which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of 'mutated clones' by a simple restriction digest.

Conclusions: The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs.

Original languageEnglish
Article number105
Number of pages9
JournalBMC Bioinformatics
Volume14
DOIs
Publication statusPublished - 22 Mar 2013

Keywords

  • Site-directed mutagenesis
  • SDM
  • PCR
  • Cloning
  • Restriction endonucleases
  • Oligonucleotides
  • Software
  • DUPLEX STABILITY
  • K+ CHANNEL
  • DNA
  • PROTOCOL
  • ARABIDOPSIS
  • EXPRESSION
  • SEQUENCES
  • INSERTION
  • DELETION

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