Primer extension based method employing nucleotides labelled...

C - Chemistry – Metallurgy – 12 – Q

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C12Q 1/68 (2006.01)

Patent

CA 2481495

The present invention relates to a method for determining the sequence of a nucleic acid molecule comprising the steps of: a) providing a single-stranded form of said nucleic acid molecule; b) hybridizing a primer to said single stranded form of said nucleic acid molecule to form a template/primer complex; c) enzymatically extending the primer by the addition of a polymerase and a mixture of at least one nucleotide and at least one labeled derivative of the at least one nucleotide, wherein the at least one labeled derivative of the at least one nucleotide comprises a label linked to the nucleotide via a cleavable link and wherein the amount of labeled derivative of the at least one nucleotide in said mixture of the at least one nucleotide and the labeled derivative of the at least one nucleotide is within the range of 1-50 mole-%, 1-40 mole-%, 1-30 mole-%, or 1-20 mole-%. d) determining the type of nucleotide added to the primer; and e) repeating steps c) to d) at least once.

La présente invention concerne un procédé permettant de déterminer la séquence d'une molécule d'acide nucléique, comprenant les étapes consistant: a) à fournir une forme simple brin de cette molécule d'acide nucléique; b) à hybrider une amorce à ladite forme simple brin de la molécule d'acide nucléique de manière à former un complexe matrice/amorce; c) à étendre par action enzymatique l'amorce par adjonction d'une polymérase et d'un mélange constitué d'au moins un nucléotide et au moins un dérivé marqué de ce nucléotide; ledit dérivé marqué comprenant un marqueur lié au nucléotide par l'intermédiaire d'une liaison pouvant être segmentée; la quantité de dérivé marqué dudit nucléotide dans le mélange susmentionné étant comprise entre 1 et 50 moles pour cent, 1 et 40 moles pour cent, 1 et 30 moles %, ou 1 et 20 mole pour cent; d) à déterminer le type de nucléotide ajouté à l'amorce; et e) à répéter les étapes c) à d) au moins une fois.

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