Nucleic acid sequence amplification method

A method of preparing a nucleic acid population comprising an amplification product maintaining a relative relationship of gene expression level in a biological sample, which method comprising

• (a) a step of amplifying a double-stranded DNA comprising a sense strand and an antisense strand, wherein the double-stranded DNA is constituted of (1) any additional nucleic acid sequence X, (2) poly T sequence, (3) cDNA sequence prepared using mRNA sequence isolated from a biological sample as a template, (4) poly A sequence and (5) any additional nucleic acid sequence Yin this order in the orientation from 5′ to 3′ of the sense strand by using the double-stranded DNA as a template, a first primer comprising any additional nucleic acid sequence X having amine added to the 5′-terminus, and optionally further comprising a poly T sequence at the downstream thereof, and a second primer comprising any additional nucleic acid sequence Y, and optionally further comprising a poly T sequence at the downstream thereof, wherein the additional nucleic acid sequence X is different from the additional nucleic acid sequence Y, such that the first primer cannot hybridize to the 3′-terminus of the sense strand of the double-stranded DNA,
• (b) a step of fragmenting the double-stranded DNA obtained in step (a),
• (c) a step of phosphorylating the 5′-termini of the fragmented double-stranded DNA obtained in step (b),
• (d) a step of preparing cDNA by using the double-stranded DNA obtained in step (c) and having a phosphorylated 5′-termini as a template, and a third primer comprising any additional nucleic acid sequence Z and said additional nucleic acid sequence Y in this order, and optionally further comprising a poly T sequence at the downstream thereof, and adding adenine (A) to the 3′-termini of the cDNA,
• (e) a step of linking a double-stranded DNA containing any sequence V having 3′-overhang thymine (T) to the double-stranded DNA obtained in step (d), and
• (f) a step of amplifying the double-stranded DNA by using the double-stranded DNA obtained in step (e) as a template, a fourth primer comprising the sequence V, and a fifth primer comprising the additional nucleic acid sequence Z, and optionally further comprising the additional nucleic acid sequence Y downstream thereof,
• thereby selectively amplifying a fragment of the double stranded DNA comprising the 3′-terminus of the sense strand of the cDNA generated in step (b).