Patent ID: 7195895
Filing Date: 2007-03-27
Classification: C07K,C12N,C12Q

Abstract:
1. A method of producing an amplified DNA molecule comprising i) amplifying a gene segment of interest by polymerase chain reaction, using a reaction solution comprising: a template comprising a first double-stranded or single-stranded DNA fragment cloned in a vector, said DNA fragment comprising a sequence encoding a protein or a portion thereof; a first sense primer that anneals with a 5′ terminal region of the protein-coding sequence; 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 a first anti-sense primer which anneals with a portion of the vector sequence downstream from the protein-coding region; thereby obtaining a first amplified DNA fragment; and ii) amplifying the first amplified DNA fragment by polymerase chain reaction, 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 hybridizing 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 hybridizing with the 3′ terminal region of the first amplified DNA fragment; b) a third sense primer which anneals with the 5′ terminal region of the second DNA fragment; and c) a second anti-sense primer which anneals with the 3′ terminal region of the third DNA fragment; wherein the DNA fragment ab) comprises regulatory sequences for transcription and translation of a gene, and the concentrations of the DNA fragments ab) and ac) in the reaction solution ii) each range from 5 to 2,500 pmol/L, thereby obtaining an amplified DNA molecule comprising the overlapped DNA fragments aa), ab) and ac).