[8] Chemical synthesis and cloning of a tyrosine tRNA gene
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Abstract:

Publisher SummaryThis chapter describes the methods used for the construction of a biologically functional suppressor transfer ribonucleic acid (tRNA) gene. For the total synthesis of given deoxyribonucleic acid (DNA)-containing biologically specific sequences, the DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands. All segments are chemically synthesized, starting with protected nucleosides and mononucleotides. The 5′-hydroxyl groups of the appropriate oligonucleotides are then phosphorylated. The chapter describes the chemical synthesis and cloning of a tyrosine tRNA gene, which involve (1) the synthesis of 126-nucleotide long bihelical DNA corresponding to a known precursor of the tyrosine suppressor tRNA, (2) sequencing of the promoter region and the distal region adjoining the C–C–A end that contains a signal for the processing of the RNA transcript (3) a total synthesis of the 208-base-pair-long DNA, which includes the control elements as well as the EcoRI restriction-endonuclease-specific sequences at the two ends, and (4) full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene. The chapter discusses a chemical synthesis of deoxyribooligonucleotides and describes the synthesis of di-, tri-, and tetranucleotides carrying 5′-phosphate groups.

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