| 摘要 |
Preparing double-stranded DNA (II), comprising a nucleic acid fragment (I), and 5' to (I) a labeling nucleic acid (L1), complementary to the 5'-end of a strand of a cleaved vector, and 3' to (I), a second labeling nucleic acid (L2), complementary to the 3' end of the same strand of the cleaved vector, is new. (II) and the vector are denatured and incubated together under hybridization conditions. Preparing double-stranded DNA (II), comprising a nucleic acid fragment (I), and 5' to (I) a labeling nucleic acid (L1), complementary to the 5'-end of a strand of a cleaved vector, and 3' to (I), a second labeling nucleic acid (L2), complementary to the 3' end of the same strand of the cleaved vector, is new. (II) and the vector are denatured and incubated together under hybridization conditions. Then copies are made of: (a) the cleaved vector, in the 5' to 3' direction, starting from the 3' end of L2; and (b) (II) in the 5' to 3' direction, starting from the 3' end of the cleaved vector. The 3' end of the strand newly synthesized using the vector as a template is ligated with the 5' end of L1, and hybridized to the 5' end of the cleaved vector. The 3' end of the strand newly synthesized using (I) as a template is ligated with the 5' end of the vector. Optionally, the entire procedure from denaturation is repeated and host cells may be transformed to select vectors that have integrated (I). Independent claims are also included for the following: (1) kits for performing the novel method; (2) an oligonucleotide (ON) polymerase chain reaction (PCR) primer for (II) comprising a 3' region homologous with one end of (I), and a 5' sequence comprising a label and having a sequence homologous with one end of the cleaved vector; (3) a pair of primers for PCR amplification of (I) comprising two primers of (2); and (4) a PCR amplification product formed using the primer pair of (3). |
