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Thermal elution of oligonucleotides on cellulose columns containing oligodeoxyribonucleotides of defined length and sequence Astell, Caroline Ruth

Abstract

The high degree of specificity of base interactions of complementary polynucleotides is a fundamental property of biological systems. Through interactions of this type, genetic information is able to flow, to succeeding generations by replication of the genome, and from deoxyribonucleic acid and ribonucleic acids to proteins, by the steps of transcription and translation. The experiments reported in this thesis are concerned with a study of the feasibility of developing a method suitable for the isolation of naturally occurring polynucleotides by hybridization of the polynucleotide with a complementary oligodeoxyribonucleotide. Homo-oligodeoxyribonucleotides (lengths up to the dodecanucleotides) of thymidylic and deoxy-adenylic acids have been prepared by chemical procedures, as have oligodeoxyribonucleotides of the repeating, complementary trinucleotide base sequences, d(pTpTpC)[sub n], d(pCpTpT)[sub n], and d(pApApG)[sub n], (n = 1 to 4). Various oligonucleotides have been linked covalently to cellulose, an insoluble, inert matrix, via the 5'phosphoryl group on the oligodeoxyribonuc1eotides, using the water-soluble carbodiimide, N-cyclohexyl-N'β(4-methylmorpholinium)ethylcarbodiimide p-toluenesulfonate. The resultant oligonucleotide-celluloses, in the form of small columns, were examined for their ability to retain complementary oli gomers. The retained oligonucleotides can be eluted conveniently with a linear temperature gradient. The data indicate that at least all but one nucleotide, and possibly the entire oligonucleotide attached to the cellulose is capable of hydrogen bonding with its complementary sequence. The resolution obtained with these columns is such that oligonucleotides, differing in length by one nucleotide residue may be fractionated. The capacity is 1/3 to 1/4 of the theoretical amount. Preliminary experiments suggest that these oligonucleotide-celluloses are capable of selectively removing a complementary sequence of nucleic acid from a mixture of nucleic acids.

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