| 摘要 |
A process according to any preceding claim for producing a double-stranded DNA construct having a nucleotide base sequence which differs at a predetermined locus from the base sequence of a starting fragment which process comprises the steps of (a) digesting and blunt ending a first sample of starting fragments having a protected 5'-terminal so as to produce a population of fragments including at least one upstream fragment; (b) ligating to the 3'-terminals of fragments produced according to step (a), first precursors for a linker; (c) digesting and blunt-ending a second sample of starting fragments having a protected 3'-terminal so as to produce a population of fragments including at least one downstream fragment; (d) ligating to the 5'-terminal of fragments produced according to step (c), second precursors for the linker; (e) ligating fragments produced according to steps (b) and (d) so as to produce a population of fragments each comprising a linker; (f) obtaining from a population of fragments produced according to step (e) at least one intermediate fragment having the structure: 5'....upstream fragment:linker:downstream fragment....3'; by probing for the upstream fragment:linker and linker:downstream fragment junctions; (g) excising at least the entire linker from an intermediate fragment produced according to step (f); and (h) ligating together the residues of upstream fragments and downstream fragments produced according to step (g), optionally with insertion of an intervening oligonucleotide, so as to produce the double-stranded DNA construct required. |
