| 摘要 |
The present invention provides a method of detecting nucleotide variation within a first nucleic acid, comprising generating a set of single-stranded extension products from a first nucleic acid in the presence of modified nucleotide bases, wherein the extension products incorporate modified nucleotides and thereby limit exonuclease activity to the 3'-terminal nucleotide base, and wherein the extension products have variable lengths, hybridizing the variable length extension products to a reference nucleic acid, contacting the hybridizing nucleic acids with an enzyme which can remove and replace the 3'-terminal nucleotide of the extension products in the presence of selected labeled nucleotides, wherein extension products that terminate with a 3'-nucleotide that does not hybridize with the corresponding position on the reference nucleic acid are replaced with one or more nucleotides that hybridize with the corresponding nucleotides on the reference nucleic acid and wherein those extension products that had a non-hybridizing nucleotide at the 3'-terminus can now be distinguished from those extension products that had a hybridizing nucleotide at the 3'-terminus, and distinguishing those extension products that had a non-hybridizing nucleotide at their 3'-terminus from those extension products that had a hybridizing nucleotide at their 3'-terminus, thereby detecting nucleotide variation in the first nucleic acid. Alternatives of this method are also provided which can also detect mutations in a nucleic acid at the penultimate 3'-terminal position on the single-stranded extension products.
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