Long PCR facilitates concise cloning and sequencing with a minimal tiling set of templates.
We demonstrate a rapid cloning and sequencing strategy for kilobase-size DNA segments using DNase I and long PCR. In a single-tube protocol, deletions were formed in a plasmid insert by two enzymatic cuts, one at a fixed site and one at random. The doubly cut molecules were recircularized to generate a library of plasmids carrying deletions of various sizes and transformed into E. coli. The plasmid inserts were directly amplified from transformant colonies by long PCR and sized on a high-resolution agarose gel. A minimal tiling set, selected from the amplified material, was used directly as templates for long-read sequencing. The system is useful for inserts up to about 3.5 kb for de novo sequencing (both strands) or 6 kb for confirmatory sequencing (one strand).
Burland
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V
FMC BioProducts, Rockland, ME, USA.
Kusukawa
N
N
D003001:Cloning, Molecular / Q000379:methods*; D003850:Deoxyribonuclease I / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D010957:Plasmids / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods; D013698:Templates, Genetic
D003850:Deoxyribonuclease I