Patent Portfolio

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The present invention provides a method for preparing a cDNA population suitable for mRNA quantification using Next Generation Sequencer, especially for a single-cell analysis, comprising (a) a step for, by using as a template a double-strand DNA configured in the order of an arbitrarily-defined additional nucleic acid sequence X, a poly-T sequence, a mRNA sequence isolated from a biological sample, a poly-A sequence, and an arbitrarily-defined additional nucleic acid sequence Y, amplifying the double-strand DNA by using a first primer comprising the arbitrarily-defined additional nucleic acid sequence X (and the poly-T sequence) having added to the 5'-end thereof an amine and a second primer comprising the arbitrarily-defined additional nucleic acid sequence Y (and the poly-T sequence), (b) a step for fragmenting the double-strand DNA obtained from step (a), (c) a step for phosphorylating the 5'-end of the fragmented double-strand DNA obtained from step (b), (d) a step for preparing a cDNA, by using as a template a double-strand DNA obtained from step (c) and of which 5'-end has been phosphorylated and a third primer comprising an arbitrarily-defined additional nucleic acid sequence Z and the additional nucleic acid sequence Y (and the poly-T sequence), and adding adenine (A) to the 3'-end of the cDNA, (e) a step for linking, to the double-strand DNA obtained from step (d), a double-strand DNA including an arbitrarily-defined sequence V having an overhang of thymine (T) at the 3'-end, and (f) a step for, by using as a template the double-strand DNA obtained from step (e) and a fourth primer including the sequence V and a fifth primer including the additional nucleic acid sequence Z (and the additional nucleic acid sequence Y), amplifying the double-strand DNA.

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