One-step insertion of oligonucleotide linkers or adapters to DNA using unphosphorylated oligonucleotides.
A simple and efficient method was developed for insertion of oligonucleotide sequences into plasmids. In this method, an unphosphorylated oligonucleotide was ligated to the restriction-digested phagemid DNA. Only the single strand of the oligonucleotide was ligated at the 5' end of the phagemid, and this resulted in the creation of a long self-complementary single-strand overhang. These single-strand overhang-possessing phagemids were used to transform XL-1 cells. This simple ligation and transformation reaction rendered approximately 7.5 x 10(4) to 5 x 10(5) of white colonies per microgram DNA from the isopropyl-beta-D-thiogalactopyranoside and 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside plate. This number is almost the same or even higher than the number of blue colonies from the control reaction in which ligase was used without the oligonucleotide. By this method we could mutate one enzyme site to another or create ribozyme and substrate phagemid very easily. Fidelity of this method was checked by restriction digestion, DNA sequencing and ribozyme reaction. By DNA sequencing, we observed that 100% of the white colonies contained a single oligonucleotide sequence.
Kang
C
C
Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Inouye
M
M
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004279:DNA, Viral / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D016337:RNA, Catalytic / Q000378:metabolism; D017422:Sequence Analysis, DNA
D004279:DNA, Viral; D009841:Oligonucleotides; D016337:RNA, Catalytic; D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII