Method for distinguishing different-sequence polynucleotides
View Patent ↗Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In the method, a mixture of sequence-specific probes are reacted with the target polynucleotide under hybridization conditions, and the hybridized probes are treated to selectively modify those probes which are bound to the target polynucleotide in a base-specific manner. The resulting labeled probes include a polymer chain which imparts to each different-sequence probe, a distinctive ratio of charge/translational frictional drag, and a detectable label. The labeled probes are fractionated by electrophoresis in a non-sieving matrix, and the presence of one or more selected sequences in the target polynucleotide are detected according to the observed electrophoretic migration rates of the labeled probes in a non-sieving medium.
1. A method of distinguishing different-sequence polynucleotides electrophoretically in a non-sieving medium, comprising
forming a plurality of different-sequence plynucleotides, each different-sequence polynucleotide containing (i) a detectable reporter label and (ii) an attached polymer chain which imparts to each different-sequence polynucleotide, a distinctive electrophoretic mobility in a non-sieving matrix,
fractionating said polynucleotides by capillary electrophoresis in a non-sieving matrix, and
detecting the fractionated polynucleotides.
2. The method of claim 1 , wherein the polymer chains have substantially the same lengths, and said different-sequence polynucleotides have different lengths.
3. The method of claim 2 , for sequencing DNA by dideoxy chain termination, wherein said different-sequence polynucleotides are formed using a 5′-primer to which said polymer chain is covalently bound.
4. The method of claim 3 , wherein said different-sequence polynucleotides terminate at their 3′-ends with dideoxynucleotides that are covalently labeled with spectrally resolvable dyes effective to distinguish the 3′-terminal nucleotide of each different-sequence polynucleotide.