Patent ID: 7846694

Claim:
A method of producing an amplified DNA fragment comprising: i) amplifying a linear double-stranded or single-stranded DNA by polymerase chain reaction (PCR), using a reaction solution comprising: a template DNA fragment comprising a double-stranded or single-stranded DNA fragment comprising a sequence encoding a protein or a portion thereof; a first sense primer that anneals with the 5′ terminal region of the template DNA fragment; a second sense primer which has a 3′ terminal sequence that is the same as at least a 5′ portion of the first sense primer and a 5′ terminal sequence that is the same as a desired nucleotide sequence; and an anti-sense primer which anneals with the 3′ terminal region of the template DNA fragment; thereby obtaining a first amplified DNA fragment; ii) amplifying the first amplified DNA fragment by polymerase chain reaction (PCR), using a reaction solution comprising: a) a template mixture comprising aa) the first amplified DNA fragment, ab) a second double-stranded or single-stranded DNA fragment comprising a sequence overlapping with the 5′ terminal region of the first amplified DNA fragment, and ac) a third double-stranded or single-stranded DNA fragment comprising a sequence overlapping with the 3′ terminal region of the first amplified DNA fragment; b) a sense primer which anneals with the 5′ terminal region of the second DNA fragment; and c) an anti-sense primer which anneals with the 3′ terminal region of the third DNA fragment; wherein the second DNA fragment comprises regulatory sequences for transcription and translation of a gene, and wherein the DNA fragment ac) has a 3′-terminal sequence that is the complement of the 5′ terminal sequence of the DNA fragment ab) and the sense primer b) is the same as the anti-sense primer c); thereby obtaining an amplified DNA molecule comprising the overlapped DNA fragments aa), ab) and ac).