Single-step hybridization screening for recombinant DNA clones with correct insert orientation and intact junction using a junctional oligonucleotide probe.
Cloning of DNA fragments with blunt ends or identical protruding ends results in colonies with the insertion sequence existing in two possible orientations. The orientation and the junctional sequence of the positive clones need to be established by means of restriction analysis and/or sequencing. Here, we proposed a rapid one-step method for the screening of clones not only with the desired orientation, but also with an intact junctional sequence. In this method, a 16-18 meric oligonucleotide probe synthesized according to the expected vector-insert junctional sequence is used as a probe in colony hybridization screening. Using this strategy, only recombinant clones which fulfill the above criteria will show positive signals in hybridization.
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Department of Medical Research, National Yang-Ming University Taipei, Taiwan, Republic of China.
Lo
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Wang
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Choo
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KB
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004274:DNA, Recombinant / Q000032:analysis*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli; D005814:Genes, Viral; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods
D017931:DNA Primers; D004274:DNA, Recombinant; D019309:Epstein-Barr Virus Nuclear Antigens; D015345:Oligonucleotide Probes