US 5,352,578 AGranted

Method of separating oligonucleotides from a mixture

AssigneeWorcester Foundation for Experimental Biology

InventorsSudhir Agrawal, Paul C. Zamecnik

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Abstract

A method of purifying full length synthetic target oligonucleotides from a mixture of oligonucleotides, particularly from a mixture containing truncated or failed sequences is disclosed. The method involves attaching a short nucleotide sequence complementary to the 5' end of a target oligonucleotide to a solid support. The complementarity between the most 5' nucleotides of the target oligonucleotide and the bound oligonucleotide results in hybridization which serves to retain the target oligonucleotide. Truncated or failed sequences lacking 5' sequences complementary to the attached oligonucleotide, fail to hybridize and therefore are not retained. The method makes it possible to purify gram quantities of synthetic deoxyribonucleic acids or ribonucleic acids and sequences which have modifications, such as on the phosphate backbone. The support-bound nucleotide sequences are stable under conditions of purification and therefore can be reused.

Claims9

Dependent claims

A method of separating full length target oligonucleotides from failed sequences in an oligonucleotide mixture resulting from an oligonucleotide synthesis reaction, the method comprising the steps of:

a. covalently binding short oligonucleotides having nucleotide sequences complementary to the 5' end of the full length target oligonucleotides to a solid support, thereby producing covalently bound oligonucleotides;
b. contacting the covalently bound oligonucleotides and the oligonucleotide mixture resulting from an oligonucleotide synthesis reaction under conditions appropriate for hybridization of complementary nucleotide sequences resulting in hybridization of covalently bound oligonucleotides with full length target oligonucleotides and with nearly full length failed oligonucleotides;
c. subjecting the hybridized products of (b) to a gradient appropriate for disrupting the hybridization of covalently bound oligonucleotides with nearly full length failed oligonucleotides, but not for disrupting the hybridization of covalently bound oligonucleotides with full length target oligonucleotides;
d. recovering full length target oligonucleotides by subjecting the covalently bound oligonucleotide-target oligonucleotide products remaining in (c) to conditions appropriate for disrupting by hybridization.

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Dependencies are inferred from the claim text.