Method for distinguishing different-sequence polynucleotides
View Patent ↗Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In one embodiment of the invention, a plurality of different-sequence probe pairs are added to a target polynucleotide, where each probe pair includes two polynucleotide probe elements which are complementary in sequence to adjacent portions of a selected one of the target sequences in the target polynucleotide. In each probe pair, one of the probe elements contains a non-polynucleotide polymer chain which imparts a distinctive mobility to the associated probe pair, when the elements in the pair are ligated. The other element in the pair contains a detectable reporter label. After the probe pairs have been allowed to hybridize with the target polynucleotide, the hybridized polynucleotides are treated under conditions effective to ligate the end subunits of target-bound probe elements when their end subunits are base-paired with adjacent target bases. The ligated probe pairs are then released from the target polynucleotide and separated electrophoretically in a sieving matrix, or chromatographically.
1. A method of distinguishing different-sequence polynucleotides electrophoretically in a sieving medium, comprising
forming a plurality of different-sequence polynucleotides, 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 sieving matrix,
fractionating said polynucleotides by capillary electrophoresis in a 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.