Total synthesis of a gene.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biochemistry

Background:

• Chemical synthesis of DNA is crucial for genetic engineering and synthetic biology.
• Previous methods faced limitations in creating long, biologically specific DNA sequences.
• The need for precise control over DNA sequences for functional gene synthesis is paramount.

Purpose of the Study:

• To develop a method for the total synthesis of DNA with biologically specific sequences.
• To construct a biologically functional suppressor transfer RNA gene.
• To characterize the synthesized gene for biological activity.

Main Methods:

• Chemically synthesizing short, overlapping single-stranded DNA segments.
• Phosphorylating oligonucleotide ends using [γ-32P]ATP and polynucleotide kinase.
• Joining segments into duplexes using polynucleotide ligase and heat-to-tail joining.

Main Results:

• Successful synthesis of a 126-nucleotide DNA precursor for tyrosine suppressor transfer RNA.
• Sequencing of promoter and C-C-A terminal regions for RNA processing signals.
• Total synthesis of a 207 base-pair DNA gene with control elements and restriction sites.
• In vitro transcription and in vivo activity confirmed the functional suppressor transfer RNA gene.

Conclusions:

• The developed method allows for the precise and efficient total synthesis of functional DNA molecules.
• This technique is applicable to the construction of complex genetic elements, such as tRNA genes.
• The synthesized gene demonstrated biological activity, validating the synthetic approach.