Ligation-mediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest

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Abstract
A method is described which permits the ligationmediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest. Feasibility was tested using BclI and phage λ DNA as a model enzyme and amplicon system, respectively. BclI is one of many widely used restriction enzymes which cleave at palindromic recognition sequences and leave 5′-protruding ends of defined sequence. Using a single pair of universal primers, a given fragment can be specifically amplified after joining the fragments to adaptors consisting of a duplex primer region and a 9-nucleotide protruding single-stranded 5′-end containing the sequence complementary to the cleaved restriction site and a 4-nucleotide ‘indexing sequence.’ The protruding strand anneals to a restriction fragment by displacing its corresponding strand in the same fragment-specific indexing sequence located juxtaposed to the restriction site. The adaptor is covalently linked to the restriction fragment by T4 DNA ligase, and amplification is carried out under conditions for long-distance PCR using the M13 forward and reverse primers. The technique discriminated robustly between mismatches and perfect matches for the 16 indexing sequences tested to allow individual λ BclI fragments to be amplified from their respective adaptor pairs. A strategy is proposed enabling a non-cloning approach to the accession, physical mapping and sequencing of genomic DNA. The method could also have application in high-throughput genetic mapping and fingerprinting and should expand the enzyme base for ligationmediated indexing technology which has previously been limited to the Class-IIS and IP restriction endonucleases.

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