Generic SBE-FRET protocol

A method of determining the identity of one or more nucleotides of interest in more than one nucleic acid sequence of interest, comprising the steps of;

• a) combining
• i) more than one nucleic acid sequence of interest, each having at least one nucleotide of interest,
• ii) a detection probe comprising a detection sequence and a donor fluorescent molecule,
• iii) one or more primers comprising a variable nucleic acid sequence which hybridizes to one of said nucleic acid-sequences of interest immediately adjacent to a nucleotide of interest and a constant region comprising a nucleic acid sequence complementary to said detection sequence of the detection probe, and
• iv) one or more dideoxynucleotides labeled with an acceptor fluorescent molecule, under conditions suitable for template-dependent single base extension of said primers, wherein said acceptor fluorescent molecule-labeled dideoxynucleotides are added to said primers by template-directed extension, thereby forming one or more detection complexes comprising a primer extended by one labeled dideoxynucleotide and a detection probe, wherein the detection probe is hybridized to the constant region of said extended primer;
• and
• b) detecting one or more of said detection complexes of a), thereby determining the identity of one or more nucleotides of interest in at least one nucleic acid sequence of interest.

A method of determining the identity of one or more nucleotides of interest in at least one nucleic acid sequence of interest comprising the steps of;

• a) combining
• i) a nucleic acid sequence of interest having at least one nucleotide of interest,
• ii) a detection probe comprising a detection sequence and a donor fluorescent molecule,
• iii) a primer comprising a variable nucleic acid sequence which hybridizes to said nucleic acid sequence of interest immediately adjacent to said nucleotide of interest, and a constant region comprising a guanine/cytosine-rich nucleic acid sequence complementary to said detection sequence of the detection probe, wherein the constant region comprises SEQ ID NO:
• 2, and wherein the primer is from about 30 to about 60 nucleotides in length, and
• iv) one or more dideoxynucleotides labeled with an acceptor fluorescent molecule, under conditions suitable for template-dependent single base extension of said primer, wherein said dideoxynucleotide is added to said primer by template-directed extension, thereby forming a detection complex;
• and
• b) detecting the detection complex of a).

A method of determining the identity of one or more nucleotides of interest in at least one nucleic acid sequence of interest comprising the steps of;

• a) combining
• i) a nucleic acid sequence of interest having at least one nucleotide of interest,
• ii) a detection probe comprising a guanine/cytosine-rich detection sequence and a donor fluorescent molecule, wherein the detection sequence is from about 10 to about 40 nucleotides in length and wherein the detection sequence comprises SEQ ID NO:
• 1,
• iii) a primer comprising a variable nucleic acid sequence which hybridizes to said nucleic acid sequence of interest immediately adjacent to said nucleotide of interest, and a constant region comprising a nucleic acid sequence complementary to said detection sequence of the detection probe, and
• iv) one or more dideoxynucleotides labeled with an acceptor fluorescent molecule, under conditions suitable for template-dependent single base extension of said primer, wherein said dideoxynucleotide is added to said primer by template-directed extension, thereby forming a detection complex;
• and
• b) detecting the detection complex of a).

A method of determining the identity of one or more nucleotides of interest in more than one nucleic acid sequence of interest, comprising the steps of;

• a) combining
• i) more than one nucleic acid sequence of interest, each having at least one nucleotide of interest,
• ii) a detection probe comprising a detection sequence and a donor fluorescent molecule,
• iii) one or more primers comprising a variable nucleic acid sequence which hybridizes to one of said nucleic acid sequences of interest immediately adjacent to a nucleotide of interest and a constant region comprising a guanine/cytosine-rich nucleic acid sequence complementary to said detection sequence of the detection probe, wherein the constant region comprises SEQ ID NO:
• 2 and wherein the primers are from about 30 to about 60 nucleotides in length, and
• iv) one or more dideoxynucleotides labeled with an acceptor fluorescent molecule, under conditions suitable for template-dependent single base extension of said primers, wherein said acceptor fluorescent molecule-labeled dideoxynucleotides are added to said primers, by template-directed extension, thereby forming one or more detection complexes;
• b) detecting one or more of said detection complexes of a).

A method of determining the identity of one or more nucleotides of interest in more than one nucleic acid sequence of interest, comprising the steps of;

• a) combining
• i) more than one nucleic acid sequence of interest, each having at least one nucleotide of interest,
• ii) a detection probe comprising a guanine/cytosine-rich detection sequence and a donor fluorescent molecule, wherein the detection sequence is from about 10 to about 40 nucleotides in length and wherein the detection sequence comprises SEQ ID NO:
• 1,
• iii) one or more primers comprising a variable nucleic acid sequence which hybridizes to one of said nucleic acid sequences of interest immediately adjacent to a nucleotide of interest and a constant region comprising a nucleic acid sequence complementary to said detection sequence of the detection probe, and
• iv) one or more dideoxynucleotides labeled with an acceptor fluorescent molecule, under conditions suitable for template-dependent single base extension of said primers, wherein said dideoxynucleotides are added to said primers by template-directed extension, thereby forming one or more detection complexes;
• b) detecting one or more of said detection complexes of a).