Ligation independent cloning irrespective of restriction site compatibility.
Here we report the use of exonuclease to expose complementary DNA between an insert and vector such that annealing becomes independent of restriction site compatibility. We demonstrate that unusual and, in some cases, previously impossible cloning strategies can be readily and efficiently achieved as long as the flanking sequences of the linear vectors are highly related. Furthermore, we show that the bacterial repair system resolves the residual mismatches, overhangs or gaps in a predictable fashion to generate excisable inserts. This approach facilitates cloning regardless of restriction site compatibility and overcomes an important limitation in current cloning techniques.
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Gene Expression Laboratory, The Salk Institute for Biological Studies, Howard Hughes Medical Institute, 10010 North Torrey Pines Road, La Jolla, CA 92186, USA. cli@axpl.salk.edu
Evans
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D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D014764:Viral Proteins / Q000378:metabolism
D018076:DNA, Complementary; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific