| 主权项 |
1. A method of copying a polynucleotide template, the method comprising:
(A) annealing a nucleic acid comprising the polynucleotide template to a first primer, wherein the polynucleotide template comprises in the 5′ to 3′ direction a second nucleotide sequence and a first nucleotide sequence; and the first primer comprises in the 5′ to 3′ direction a first nucleotide sequence and a second nucleotide sequence, wherein the second nucleotide sequence of the first primer comprises a nucleotide sequence which is complementary to the first nucleotide sequence of the polynucleotide template; (B) synthesizing a first extension product from the first primer in the presence of a polymerase; (C) annealing a second primer to the first extension product, wherein the second primer comprises in the 5′ to 3′ direction a first nucleotide sequence and a second nucleotide sequence, wherein the second nucleotide sequence of the second primer comprises a nucleotide sequence which is complementary to a partner nucleotide sequence, wherein the partner nucleotide sequence is a nucleotide sequence complementary to the second nucleotide sequence of the polynucleotide template; (D) synthesizing a second extension product from the second primer in the presence of the polymerase; (E) annealing the first primer to the 3′ end of the second extension product and synthesizing a third expression product from the first primer in the presence of the polymerase; (F) generating a double-stranded nucleic acid comprising the third extension product and the second extension product; (G) repeating at least steps (E)-(F) one or more additional times to generate at least a first copy and a second copy of the double-stranded nucleic acid comprising the third extension product and the second extension product; (H) annealing the 3′ terminal region of the third extension product from the first copy of the double-stranded nucleic acid to the 3′ terminal region of the second extension product from the second copy of the double-stranded nucleic acid to produce a cross-over structure; (I) synthesizing a fourth extension product from the third extension product of step (H) and a fifth extension product from the second extension product of step (H) in the presence of the polymerase; and (J) producing a concatemer comprising at least two copies of the polynucleotide template, wherein the concatemer comprises the fourth extension product and the fifth extension product, and wherein the concatemer comprises a first concatemer strand, wherein the first concatemer strand comprises a 5′ end and a 3′ end, and comprises a nucleotide sequence having the general structure in the 5′ to 3′ direction of: C′-T-C′-T-C′, wherein: C′ is the first nucleotide sequence of the second primer, and T is a nucleotide sequence of the polynucleotide template; and wherein steps (A)-(J) are performed in a reaction mixture at a substantially isothermal temperature and wherein the reaction mixture does not comprise a recombinase enzyme. |
