Nucleic Acid Analysis Using Non-templated Nucleotide Addition
One embodiment of the invention is a method of producing an oligonucleotide extended by a single nucleotide base. An oligonucleotide and an extension terminating nucleotide are mixed with an enzyme having terminal transferase activity. The reaction produces an oligonucleotide extended by a single base. The extended oligonucleotide may be used as a size standard for single base extension reactions. Another embodiment of the invention is a method of producing a mixture of oligonucleotides extended by different single bases. An oligonucleotide, a first extension terminating nucleotide, and a second extension terminating nucleotide are mixed with an enzyme having terminal transferase activity. The first and second extension terminating nucleotides comprise different nucleotide bases and are labeled with different labels. The identity of the different extension terminating nucleotides (and hence the extended oligonucleotides) may be ascertained by reference to the specific label incorporated.
1 - 13 . (canceled)
14 . A method of identifying the reaction product of a single nucleotide base extension reaction on a detection instrument, said method comprising,
forming a single base oligonucleotide extension product standard by mixing an oligonucleotide with an extension terminating nucleotide and a terminal transferase, and
resolving the single base oligonucleotide extension product standard on a detection instrument, whereby a signal indicative of the single base oligonucleotide extension product standard is created.
15 . The method of claim 14 , comprising,
forming a single base extension reaction product standard by mixing a second oligonucleotide with an extension terminating nucleotide and an enzyme having DNA polymerase activity,
resolving the single base extension product on the detection instrument, whereby a signal indicative of the single base extension product is created,
comparing the signal indicative of the single base oligonucleotide extension product with the signal indicative of the single base extension product.
16 - 17 . (canceled)
18 . A method of identifying the reaction product of a single nucleotide base extension reaction on a detection instrument, said method comprising,
forming a single base oligonucleotide extension product standard by mixing an oligonucleotide with an extension terminating nucleotide and an enzyme having terminal transferase activity, wherein the mixture does not contain a template that permits the oligonucleotide to function as a primer, and
resolving the single base oligonucleotide extension product standard on a detection instrument, whereby a signal indicative of the single base oligonucleotide extension product standard is created.