Method for Gene Amplification

Abstract

The present invention provides a double-stranded DNA constructed specifically for high speed gene amplification, a method for gene amplification and a method for synthesizing protein. The gene amplification system of the present invention used a site-specific recombinase such as Cre-lox system and target sequence thereof to efficiently induce a type of replication referred to as a double rolling-circle replication (DRCR). Amplification unit, whose structure is shown in FIG. 2 ( a ), is constructed in animal and other cells. DRCR is induced by two recombination events triggered by a site-specific recombinase (Cre) when each replication folk progresses between each pair of target sequences (lox sequences).

1 . A double-stranded DNA represented by a-b-c-d or a-c-b-d, wherein one of a and b is a double-stranded DNA fragment comprising a first target sequence of a site-specific recombinase, and the other is a double-stranded DNA fragment comprising an inverted sequence of said first target sequence; and one of c and d is a double-stranded DNA fragment comprising a second target sequence of the site-specific recombinase and the other is a double-stranded DNA fragment comprising an inverted sequence of said second target sequence; a replication origin and at least one target gene to be amplified are inserted anywhere between a and d; and arbitrary DNA sequences may be inserted among above fragments. 
     
     
         2 . The double-stranded DNA of  claim 1 , wherein b and care combined and said double-stranded DNA is represented by a-b-d, wherein a and d are the same sequence with the same direction and the other letters are the same as defined previously. 
     
     
         3 . The double-stranded DNA of  claim 1 , which is represented by a-b-X-c-d or a-c-X-b-d, wherein X represents a replication origin and the other letters are the same as defined previously. 
     
     
         4 . The double-stranded DNA of  claim 3 , which is represented by a-A-b-X-c-B-d or a-A-c-X-b-B-d, wherein at least one of A and B represents the target gene, arbitrary DNA sequences may be inserted among these fragments, and the other letters are the same as defined previously. 
     
     
         5 . The double-stranded DNA of  claim 1 , wherein the first target sequence and the second target sequence of the site-specific recombinase are different. 
     
     
         6 . The double-stranded DNA of  claim 1 , wherein each of said the first and the second target sequences is selected from the group comprising loxP, lox511, lox5171, lox2272, lox2372, loxm2, loxFAS, lox71, lox66 and the mutants thereof in a case where the site-specific recombinase is Cre recombinase or its derivative; each of said the first and the second target sequences is selected from the group comprising FRT, F3, F5, FRT mutant−10, FRT mutant+ 10 and the mutants thereof in a case where the site-specific recombinase is Flp recombinase or its derivative; and each of said the first and the second target sequences is selected from the group comprising attB, attP and the mutants thereof 10 in a case where the site-specific recombinase is phiC31 integrase or its derivative. 
     
     
         7 . A vector comprising the double-stranded DNA of  claim 1 . 
     
     
         8 . A transformant, which is introduced with the double-stranded DNA of  claim 1 . 
     
     
         9 . The transformant of  claim 8 , wherein the host is an animal cell. 
     
     
         10 . A set of double-stranded DNA fragments, which is obtained by dividing any one of the double-stranded DNA of  claim 1  into at least two, wherein each said fragment contains a double-stranded DNA region with at least 50 bp at both ends for homologous recombination; said double-stranded DNA region for homologous recombination comprises a part of the sequences of said host chromosome or an extrachromosomal element so that the double-stranded DNA can be integrated into the host chromosome or the extrachromosomal element by homologous recombination; and said replication origin may be a replication origin of said host or said exogeneous origin. 
     
     
         11 . The set of double-stranded DNA of  claim 10  comprising a double-stranded DNA fragment represented by e-a-A-b-f and a double-stranded DNA fragment represented by g-c-B-d-h, wherein one of a and b is a double-stranded DNA fragment comprising a first target sequence of a site-specific recombinase, and the other is a double-stranded DNA fragment comprising an inverted sequence of said first target sequence; and one of c and d is a double-stranded DNA fragment comprising a second target sequence of the site-specific recombinase and the other is a double-stranded DNA fragment comprising an inverted sequence of said second target sequence; each of letters from e to h is a double-stranded DNA fragment of at least 50 bp in size, which are arranged on a chromosome or an extrachromosomal element that is a host for integration of the set of double-stranded DNA in order of e, f, a replication origin of the chromosome element or the extrachromosomal element, g and h; at least one of A and B represents the target gene to be amplified; and said replication origin or a part of it may be included in for g; and an arbitrary DNA sequence may be inserted among these. 
     
     
         12 . The set of double-stranded DNA of  claim 10 , wherein the first target sequence and the second target sequence of the site-specific recombinase are different. 
     
     
         13 . The set of double-stranded DNA of  claim 10 , wherein each of said the first and the second target sequences is selected from the group comprising loxP, lox511, lox5171, lox2272, lox2372, loxm2, loxFAS, lox71, lox66 and the mutants thereof in a case where the site-specific recombinase is Cre recombinase or its derivative; each of said the first and the second target sequences is selected from the group comprising FRT, F3, F5, FRT mutant−10, FRT mutant+10 and the mutants thereof in a case where the site-specific recombinase is Flp recombinase or its derivative; and each of said the first and the second target sequences is selected from the group comprising attB, attP and the mutants thereof in a case where the site-specific recombinase is phiC31 integrase or its derivative. 
     
     
         14 . A set of vectors, wherein each vector contains each of two kinds of the double-stranded DNA of  claim 10 . 
     
     
         15 . A transformant, which is introduced with two kinds of the double-stranded DNA of  claim 10 , wherein said replication origin locates on a host chromosome or an extrachromosome. 
     
     
         16 . The transformant of  claim 15 , wherein the host is an animal cell. 
     
     
         17 - 19 . (canceled)