Well-defined insoluble primers for the enzymatic synthesis of oligo- and polynucleotides.
Two methods are described by which primer molecules like UpU and oligodeoxythymidylates can be coupled with high efficiency to an insoluble polymer, like hydroxypropylated Sephadex G-50, by one covalent linkage. In one procedure aliphatic dicarboxylic dichlorides (e.g. adipoyl dichloride) are used to serve as spacers of variable length and for anchoring the primer molecule UpU. The other method involves pU as an anchor for (pdT)3 and (pdT)6, which are coupled to the polymer using condensation reactions with 2,4,6-triisopropylphenylsulfonyl chloride. In both cases the homogeneous primer molecules are bound specifically to the polymer. The insoluble primers are tested for their priming efficiency using polynucleotide nucleotidyltransferase from Micrococcus luteus and DNA nucleotidylexotransferase from calf thymus. The primers and synthesized polynucleotides can be cleaved from the polymer under conditions which are not damaging to ribo- and deoxyribopolynucleotides.
D000818:Animals; D002417:Cattle; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D007700:Kinetics; D008722:Methods; D008837:Micrococcus / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000378:metabolism; D009841:Oligonucleotides / Q000096:biosynthesis*; D009843:Oligoribonucleotides / Q000096:biosynthesis*; D011061:Poly A / Q000096:biosynthesis; D011131:Polyribonucleotides / Q000096:biosynthesis*; D013950:Thymus Gland / Q000201:enzymology; D014529:Uridine
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D011131:Polyribonucleotides; D011061:Poly A; D004254:DNA Nucleotidyltransferases; D009713:Nucleotidyltransferases; D014529:Uridine