Various 5'O-N-protected deoxynucleoside-3'-O-beta-cyanoethyl-N,N-dialkylamino-/N- morpholinophosphoramidites were prepared from beta-cyanoethyl monochlorophosphoramidites of N,N-dimethylamine, N,N-diisopropylamine and N-morpholine. These active deoxynucleoside phosphates have successfully been used for oligodeoxynucleotide synthesis on controlled pore glass as polymer support and are very suitable for automated DNA-synthesis due to their stability in solution. The intermediate dichloro-beta- cyanoethoxyphosphine can easily be prepared free from any PC1(3) contamination. The active monomers obtained from beta-cyanoethyl monochloro N,N- diisopropylaminophosphoramidites are favoured. Cleavage of the oligonucleotide chain from the polymer support, N-deacylation and deprotection of beta-cyanoethyl group from the phosphate triester moiety can be performed in one step with concentrated aqueous ammonia. Mixed oligodeoxynucleotides are characterized by the sequencing method of Maxam and Gilbert.
There is an increasing demand for vector-free single stranded insert DNAs for the use as probes in either filter-, solution-, or in-situ hybridization experiments. This prompted us to modify the well established M13mpl8/19 vectors (1) in a way that cloned single stranded inserts can be excised via inverted repeats forming a master restriction site upon annealing. The original M13mpl8/19 polylinker region was removed by digestion with Hind III and Eco RI and replaced by a synthetic 96 bp polylinker (Fig. Ia). The original Eco RI and Hind III sites were inactivated by replacing G by A at both ends of the new polylinker ( marked by stars; Fig la).
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