In the method, an oligonucleotide primer hybridizes to a complementary region along the nucleic acid , to form a duplex, with the primer’s terminal 3’ end directly adjacent to the nucleotide base to be identified. The oligonucleotide primer is enzymatically extended by a single base in the presence of all four nucleotide terminators; the nucleotide terminator complementary to the base in the template being interrogated is incorporated and identified. The presence of all four terminators ensures that no further extension occurs beyond the single incorporated base . Many approaches can be taken for determining the identity of a terminator, including fluorescence labeling, mass labeling for mass spectrometry, measuring enzyme activity using a protein moiety, and isotope labeling .
In the method, an oligonucleotide primer hybridizes to a complementary region along the nucleic acid to form a duplex, with the primer’s terminal 3’ -end directly adjacent to the nucleotide base to be identified. Using a DNA polymerase, the oligonucleotide primer is enzymatically extended by a single base in the presence of all four nucleotide terminators; the nucleotide terminator complementary to the base in the template being interrogated is incorporated and identified. The presence of all four terminators suppresses misincorporation of non-complementary nucleotides . Many approaches can be taken for determining the identity of an incorporated terminator, including fluorescence labeling, mass labeling for mass spectrometry, isotope labeling, and tagging the base with a hapten and detecting chromogenically with an anti-hapten antibody-enzyme conjugate (e.g., via an ELISA format) .
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end
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The
Using a DNA polymerase, the
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ensures that no further extension occurs beyond the single incorporated base
suppresses misincorporation of non-complementary nucleotides
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an incorporated
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measuring enzyme activity using a protein moiety, and isotope labeling
isotope labeling, and tagging the base with a hapten and detecting chromogenically with an anti-hapten antibody-enzyme conjugate (e.g., via an ELISA format)