Multiple site directed mutagenesis strategy based on total RNA and RT-PCR method

Zsolt B. Nagy, Márk Gárdonyi, Attila Mészáros, Zoltán Varga-Orvos, Robert G. Solomon, L. Tamás

Research output: Contribution to journalArticle

Abstract

Site-directed PCR-based mutagenesis methods are widely used to generate mutations. All published methods work on DNA clones carrying the target sequence. However, DNA clones are not always available. We have previously published a RT-PCR-based site-directed mutagenesis method starting from total RNA to overcome this problem. In this article, we report an improvement of our previous method to facilitate introduction of multiple mutations into a target sequence. We demonstrate the efficacy and feasibility of this strategy by mutation of the human β-actin gene. BamHI restriction endonuclease cleavage sites were generated within the gene to assist screening. Using three mutagenic primers in a single RT-PCR reaction, seven different clones were produced carrying three single and four multiple mutations. An investigation of the effect of the cycle number and elongation time of the PCR reactions revealed that both have an influence on the ratio of clones carrying single and multiple mutations. An optimized protocol was established for efficient multiple site-directed mutagenesis.

Original languageEnglish
Pages (from-to)206-211
Number of pages6
JournalMolecular Biotechnology
Volume37
Issue number3
DOIs
Publication statusPublished - Nov 2007

Fingerprint

Mutagenesis
Site-Directed Mutagenesis
RNA
Polymerase Chain Reaction
Clone Cells
Mutation
DNA
Genes
DNA Restriction Enzymes
Deoxyribonuclease BamHI
Actins
Elongation
Screening

Keywords

  • Megaprimer
  • Multiplex mutations
  • PCR-based strategy
  • RT-PCR
  • Site-directed mutagenesis
  • Total RNA

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Multiple site directed mutagenesis strategy based on total RNA and RT-PCR method. / Nagy, Zsolt B.; Gárdonyi, Márk; Mészáros, Attila; Varga-Orvos, Zoltán; Solomon, Robert G.; Tamás, L.

In: Molecular Biotechnology, Vol. 37, No. 3, 11.2007, p. 206-211.

Research output: Contribution to journalArticle

Nagy, ZB, Gárdonyi, M, Mészáros, A, Varga-Orvos, Z, Solomon, RG & Tamás, L 2007, 'Multiple site directed mutagenesis strategy based on total RNA and RT-PCR method', Molecular Biotechnology, vol. 37, no. 3, pp. 206-211. https://doi.org/10.1007/s12033-007-0042-0
Nagy, Zsolt B. ; Gárdonyi, Márk ; Mészáros, Attila ; Varga-Orvos, Zoltán ; Solomon, Robert G. ; Tamás, L. / Multiple site directed mutagenesis strategy based on total RNA and RT-PCR method. In: Molecular Biotechnology. 2007 ; Vol. 37, No. 3. pp. 206-211.
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