Resolution of sequencing ambiguities: a universal FokI adapter permits Maxam-Gilbert re-sequencing of single-stranded phagemid DNA.
We propose a method to resolve ambiguities encountered when single-stranded (ss) phagemid DNA templates are sequenced by the dideoxy method. A single oligodeoxyribonucleotide (oligo) is synthesized with the following features: (i) the 20 nucleotides (nt) at the 5'-end form a double-stranded hairpin containing a FokI restriction site, exactly as previously described by Podhajska and Szybalski [Gene 40 (1985) 175-182]; (ii) the 23 nt at the 3'-end hybridize to the (+)strand of ss phagemid DNA in the region complementary to the M13 universal sequencing primer. In a simple one-tube set of reactions, ss phagemid DNA is annealed to this oligo, cleaved by FokI at a unique site outside the vector multiple cloning site and then labelled at this unique site by Klenow polymerase and [alpha-32P]dCTP. These reactions provide a convenient route by which Maxam-Gilbert chemical degradation sequencing methods can be used to resolve ambiguities encountered in the dideoxy-sequencing of a unidirectional deletion series already prepared in popular phagemid vectors. A single oligo allows labelling of all members of a deletion series. A second universal oligo allows the same set of reactions to be applied to inserts cloned into (-)strand phagemids.
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
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Goszczynski
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McGhee
D000818:Animals; D001483:Base Sequence; D002107:Caenorhabditis / Q000235:genetics*; D002872:Chromosome Deletion; D004277:DNA, Single-Stranded / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D015183:Restriction Mapping; D013698:Templates, Genetic; D014780:Viruses / Q000235:genetics
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific