Nucleic acids site-directed mutagenesis

This company founded in 1994, is a contract molecular biology and screening service company for the pharmaceutical, research, and clinical markets worldwide. Custom services include gene synthesis, DNA sequencing, site-directed mutagenesis, nucleic acid purification, and genotype/genetic analysis. 

Carter, P., Bedouelle, H., and Winter, G. (1985) Improved oligonucleotide site-directed mutagenesis using ml 3 vectors Nucleic Acids Res 13,4431—4443... 

G. Cortopassi, and D. J. Gales, A simple method for site-directed mutagenesis using PCR, Nucleic Acid Res. 1989, 17, 6545-6551. 

With a small adjustment to this procedure, using thiophosphoryl chloride (PSCI3) in place of phosphoryl chloride (POCI3), the nucleoside 5 -(l-thio)triphosphates can be easily synthesized.7 These compounds have been used extensively for applications inter alia for site-directed mutagenesis, sequencing of nucleic acids and investigation of enzyme mechanisms.16 Phosphorylation with thiophosphoryl chloride is generally slower than with phosphoryl chloride but still occurs at a reasonable rate for purine nucleosides. However, for pyrimidine nucleosides, it is necessary to add 2,4,6-collidine as a catalyst, which forms a reactive intermediate with the thiophosphoryl chloride in situ. 

Callow, M. J., Perrin, L. J., and Rubin, E. M. 1994. Single base, site-directed mutagenesis of a 90 kilobase-pair PI clone. Nucleic Acids Res., 22, 4348-4349. 

Because the Pol II core alone is sufficient to maintain the transcription bubble and the DNA-RNA hybrid during RNA chain elongation, there must be exposed elements on the enzyme surface that keep the nucleic acid strands apart. Protein elements are needed to separate the DNA strands downstream of the active site and to separate the RNA from the DNA template strand at the upstream end of the hybrid. On the basis of their location with respect to nucleic acids, several Pol II structural elements are predicted to maintain the bubble and the hybrid. These proposals are currently tested by site-directed mutagenesis. Separation of the DNA strands at the downstream edge of the bubble may be attributed to binding of the DNA template strand by switch regions 1 and 2 and to blocking of the path of the nontemplate strand by fork loop 2. In the Pol II-TFIIS complex structure, fork loop 2 is ordered and restricts the cleft to a diameter of 15 A, consistent with the proposal that this loop removes the DNA nontemplate strand from the template strand before the active site. 

Friedhoff, P. Kolmes, B. Gimadutdinow, O. Wende, W. Krause, K. L. Pingoud, A., Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis. Nucleic Acids Res 1996,24,2632-9. 

Hydrolytic cleavage of N,0-acetals due to enzymatic processes leads to nucleic acid chain elements without a nucleobase. Such positions are potential mutation sites. To study such mechanisms and to induce directed mutagenesis, oligonucleotides have been synthesized containing tetrahydrofuran building blocks introduced via corresponding phosphoramidites [310,311] (Fig. 35). 

---