Separation and sequencing of the sequence isomers of pyrimidine deoxypentanucleoside tetraphosphates by high-performance liquid chromatography.

Chemical synthesis of DNA fragments with a defined sequence is extremely long and complex. A simpler alternative is isolation of fragments from partially hydrolysed DNA. Mixtures of sequence isomers of pyrimidine oligonucleotides were recently isolated on a preparative scale from hydrolysates of depurinated herring sperm DNA. These, however, could not be further separated by conventional techniques. Using high-performance liquid chromatography (HPLC), sequence isomers of pyrimidine di-, tri- and tetranucleotides were successfully separated and sequenced. In the present work, separation and sequencing of sequence isomers of pyrimidine pentanucleotides by HPLC was achieved, demonstrating the applicability of this particular approach in preparation of desired DNA fragments.