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Published on: October 23, 2017
Development of a technique for multiple site-directed mutagenesis of the ftf gene of Streptococcus salivarius
1Institute of Dental Research, United Dental Hospital of Sydney, Surry Hills, NSW, Australia.
Abstract:
Attempts at site-directed mutagenesis of the fructosyltransferase (ftf) gene of Streptococcus salivarius ATCC 25975 using standard protocols were unsuccessful and resulted in a series of deletions. These deletions appeared to commence at points within the ftf gene where there were palindromic sequences which were capable of forming closed loop structures that acted as terminators under the conditions of mutagenesis. To overcome this problem, two modified mutagenic techniques were developed. They made use of T4 DNA polymerase in conjunction with either T7 DNA polymerase at 37 degrees C or Vent DNA polymerase from Thermococcus litoralis at an elevated temperature. These methods eliminated the need for a single-stranded DNA template and allowed polymerisation through palindromic sequences to rapidly produce multiple site-directed mutations.
Insights
Modified mutagenesis techniques successfully overcame challenges in altering the fructosyltransferase (ftf) gene in Streptococcus salivarius. These new methods enable rapid, multiple site-directed mutations, advancing genetic research.
Area of Science:
- Molecular Biology
- Microbial Genetics
Background:
- Site-directed mutagenesis of the fructosyltransferase (ftf) gene in Streptococcus salivarius ATCC 25975 using standard methods resulted in deletions.
- These deletions were attributed to palindromic sequences within the ftf gene forming terminator structures during mutagenesis.
Purpose of the Study:
- To develop novel mutagenesis techniques to overcome challenges in site-directed alteration of the ftf gene.
- To enable rapid and efficient generation of multiple site-directed mutations within the ftf gene.
Main Methods:
- Development of two modified mutagenesis techniques utilizing T4 DNA polymerase.
- Employing either T7 DNA polymerase at 37°C or Vent DNA polymerase from Thermococcus litoralis at elevated temperatures.
- These methods eliminated the requirement for a single-stranded DNA template.
Main Results:
- Successful polymerization through palindromic sequences was achieved, bypassing the terminator structures.
- Rapid production of multiple site-directed mutations within the ftf gene was demonstrated.
- The modified techniques proved effective in overcoming the limitations of standard mutagenesis protocols.
Conclusions:
- The developed modified mutagenesis techniques provide a robust solution for site-directed alteration of genes containing problematic palindromic sequences.
- These methods significantly enhance the efficiency and scope of genetic engineering in Streptococcus salivarius and potentially other organisms.
- The ability to rapidly generate multiple mutations facilitates deeper understanding of gene function, such as that of the fructosyltransferase gene.
