METHODS OF SYNTHESIZING AND LABELING NUCLEIC ACID MOLECULES
The present invention is generally related to composition, kits and methods for synthesizing nucleic acid molecules and particularly for synthesizing labeled nucleic acid molecules. Specifically, the invention relates to methods, kits and compositions for synthesizing indirectly labeled nucleic acid molecules. The labeled nucleic acid molecules produced in accordance with the invention are particularly suited as labeled probes for nucleic acid detection, diagnostics, and array analysis.
1 . A method of synthesizing one or more nucleic acid molecules comprising incubating one or more nucleic acid templates with two or more modified nucleotides under conditions sufficient to make one or more first nucleic acid molecules complementary to all or a portion of said one or more templates, wherein at least one said nucleic acid molecule contains said two or more modified nucleotides incorporated therein; and
incubating said one or more nucleic acid molecules in the presence of one or more detectable labels under conditions sufficient to couple one or more of said labels to at least one of said modified nucleotides incorporated therein.
2 . A method according to claim 1 , wherein at least one of said modified nucleotides is selected from the group consisting of aminoallyl-dUTP, aminoallyl-dCTP, and aminohexyl-dATP.
3 . A method according to claim 1 , wherein at least two of said modified nucleotides is selected from the group consisting of aminoallyl-dUTP, aminoallyl-dCTP, and aminohexyl-dATP.
4 . A method according to claim 1 , wherein said nucleic acid template is mRNA or a population of mRNA molecules.
5 . A method according to claim 1 , further comprising incubating said one or more nucleic acid molecules under conditions sufficient to make one or more second nucleic acid molecules complementary to all or a portion of said one or more first nucleic acid molecules.
6 . A method according to claim 1 , wherein at least one of said labels is a fluorescent label.
7 . A method according to claim 1 , wherein at least one of said labels is a cyanine dye.
8 . A method according to claim 1 , wherein at least one of said labels is Cy3.
9 . A method according to claim 1 , wherein at least one of said labels is Cy5.
10 . A method according to claim 1 , wherein at least one of said labels is an Alexa dye.
11 . A kit comprising two or more different, modified, monomeric deoxyribonucleotide triphosphates according to claim 1 , wherein said modified deoxyribonucleotide triphosphates have the ability to bind one or more detectable labels.
12 . A kit according to claim 11 , wherein at least one of said modified nucleotides contains a reactive primary amine.
13 . A kit according to claim 11 , wherein at least 2 of said modified nucleotides contain primary reactive amines.
14 . A kit according to claim 11 , wherein at least one of said modified nucleotides is selected from the group consisting of aminoallyl-dUTP and aminohexyl-dATP.
15 . A kit according to claim 11 , wherein at least one of said modified nucleotides is aminoallyl-dUTP.
16 . A kit according to claim 11 , wherein at least one of said modified nucleotides is aminohexyl-dATP.
17 . A kit according to claim 11 , wherein at least two of said modified nucleotides is selected from the group consisting of aminoallyl-dUTP and aminohexyl-dATP.
18 . A kit according to claim 11 , further comprising at least one nucleic acid template.
19 . A kit according to claim 18 , wherein said nucleic acid template is DNA.
20 . A kit according to claim 18 , wherein said nucleic acid template is RNA.