Methods of using nucleotide analogues
The present invention provides methods, compositions, mixtures and kits utilizing deoxynucleoside triphosphates comprising a 3′-O position capped by a group comprising methylenedisulfide as a cleavable protecting group and a detectable label reversibly connected to the nucleobase of said deoxynucleoside. Such compounds provide new possibilities for future sequencing technologies, including but not limited to Sequencing by Synthesis.
1. A method for analyzing a DNA sequence comprising the steps of
a) providing a nucleic acid template with a primer hybridized to said template forming a primer/template hybridization complex,
b) adding DNA polymerase, and a first deoxynucleoside triphosphate comprising a nucleobase and a sugar selected from a mixture of at least 4 differently labeled, 3′-O methylenedisulfide capped deoxynucleoside triphosphate compounds having the structures:
c) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction so as to create a modified primer/template hybridization complex, and
d) detecting said first detectable label of said labeled 3′-O methylenedisulfide capped deoxynucleoside triphosphate in said modified primer/template hybridization complex,
e) removing said cleavable protecting group, and
f) repeating steps b) to e) at least once.
2. The method according to claim 1 , wherein the mixture of at least 4 differently labeled, 3′-O methylenedisulfide capped deoxynucleoside triphosphate compounds further comprises at least one unlabeled methylenedisulfide capped deoxynucleoside triphosphate compound, wherein the unlabeled 3′-O methylenedisulfide capped deoxynucleoside triphosphate compound is selected from: