Cloning and assembly of PCR products using modified primers and DNA repair enzymes.
We present a method for the creation of ligatable 3' overhangs by the incorporation of a modified base, uracil, at a specific position in the PCR primer and subsequent treatment with the DNA-modifying enzyme uracil DNA glycosylase and then either T4 endonuclease V or human apurinic/apyrimidinic endonuclease 1. In this study, we describe the cloning of a fragment specifying the chloramphenicol-resistance gene into a SacI vector site. To further test this method, three segments of the lacZ gene were amplified by PCR, and after treatment with the DNA-modifying enzymes, the properly oriented segments were ligated into a SacI-cleaved plasmid. Using the methods described, we were able to assemble PCR products into appropriate structures.
Watson
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Rice University, Houston, TX, USA.
Bennett
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D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002702:Chloramphenicol Resistance / Q000235:genetics; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D017931:DNA Primers; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043224:Deoxyribonuclease (Pyrimidine Dimer); D043223:Deoxyribonuclease IV (Phage T4-Induced); D015252:Deoxyribonucleases, Type II Site-Specific; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase; D014764:Viral Proteins
D017931:DNA Primers; D029968:Escherichia coli Proteins; D014764:Viral Proteins; D014498:Uracil; D015500:Chloramphenicol O-Acetyltransferase; D004706:Endodeoxyribonucleases; C024408:endodeoxyribonuclease SacI; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D015252:Deoxyribonucleases, Type II Site-Specific; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase