| 摘要 |
The present invention provides a method of nucleic acid manipulation comprising: (i) hybridizing a double stranded primer to the 3' end of a single stranded nucleic acid template, wherein said primer comprises a double stranded region and a single stranded region, wherein the single stranded region is a 3' overhang region and wherein the 3' overhang region enables the double stranded primer to target and hybridize to the 3' end of the nucleic acid template, wherein said single stranded 3' overhang region comprises a degenerate sequence or a sequence comprising universal bases, and wherein the double stranded primer is made up of two separate strands; (ii) at least one round of polymerization using a polypeptide with 5' to 3' DNA polymerization activity, wherein at least the first round of polymerization comprises using a polypeptide with 5' to 3' DNA polymerization activity to carry out a primer extension reaction to synthesise nucleotides in a template dependent manner from the 3' end of the single stranded region of said hybridized double stranded primer; wherein the hybridizing step (i) and at least the first primer extension reaction from the 3' end of the single stranded region of said hybridized double stranded primer in step (ii) takes place without the formation of a phosphodiester bond between the 3' end of the nucleic acid template and the double stranded primer of step (i). Products, kits and compositions suitable for use in such methods are also provided. |
