Direct cloning of unmodified PCR products by exploiting an engineered restriction site.
A method is described for the direct cloning of DNA fragments amplified by the polymerase chain reaction (PCR). An oligodeoxyribonucleotide, bearing two engineered XcmI sites placed in tandem, was used to generate cloning vectors bearing single 3' deoxythymidine (dT) overhangs at their ends. These 3' dT overhangs are compatible with the 3' deoxyadenosine overhangs found on most Taq polymerase-amplified PCR products. Consequently, Taq polymerase-amplified PCR products can be ligated directly into these modified restriction sites.
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Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461.
Listowsky
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Sollitti
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D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase; D003839:Deoxyadenosines / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D019914:Taq Polymerase; D013936:Thymidine / Q000737:chemistry
D003839:Deoxyadenosines; D009838:Oligodeoxyribonucleotides; D019914:Taq Polymerase; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase; C084267:endodeoxyribonuclease XcmI; D015252:Deoxyribonucleases, Type II Site-Specific; D013936:Thymidine