| 主权项 |
1. A method for amplifying a linear double-stranded nucleic acid template comprising two separate complementary strands, comprising:
(A) generating a circular strand from the linear double-stranded nucleic acid template, wherein the circular strand comprises the single-strand component of at least one restriction enzyme recognition sequence and the sequences of each of the two separate complementary strands of the template; (B) treating the circular strand with a first oligonucleotide primer and a polymerase, under conditions such that an extension product of the first primer is synthesized; (C) treating the extension product of the first primer of step (B) with a second oligonucleotide primer and a polymerase, under conditions such that an extension product of the second primer is synthesized which is complementary to the extension product of the first primer of step (B), to produce a new double-stranded nucleic acid comprising at least a portion of the extension product of the first primer and at least a portion of the extension product of the second primer; and (D) treating the new double-stranded nucleic acid of step (C) with a restriction enzyme that recognizes a full double-stranded restriction enzyme recognition sequence corresponding to the at least one restriction enzyme recognition sequence of step (A), under conditions such that the new double-stranded nucleic acid of step (C) is cleaved to form two or more shorter double-stranded nucleic acids, at least two of which shorter double-stranded nucleic acids comprise at least a portion of a copy of the linear double-stranded nucleic acid template of step (A), thereby amplifying the linear double-stranded nucleic acid template. |
