One-step insertion of oligonucleotide linkers or adapters to DNA using unphosphorylated oligonucleotides.
One-step insertion of oligonucleotide linkers or adapters to DNA using unphosphorylated oligonucleotides.
复制标题
使用未磷酸化的寡核苷酸将寡核苷酸接头或衔接子一步插入 DNA。
作者：
C. Kang;M. Inouye
建立了一种简单有效的寡核苷酸序列插入质粒的方法。在该方法中，将未磷酸化的寡核苷酸连接至限制性消化的噬菌粒DNA。仅寡核苷酸的单链连接在噬菌粒的5'末端，这导致产生长的自身互补单链突出端。这些具有单链悬突的噬菌粒用于转化XL-1细胞。这种简单的连接和转化反应使得来自异丙基-β-D-硫代半乳糖吡喃糖苷和5-溴-4-氯-3-吲哚基-β-D-半乳糖吡喃糖苷平板的每微克DNA产生约7.5 × 10（4）至5 × 10（5）个白色菌落。该数量几乎与来自对照反应的蓝色菌落的数量相同或甚至更高，在对照反应中使用连接酶而不使用寡核苷酸。通过这种方法，我们可以很容易地将一个酶位点突变为另一个酶位点或产生核酶和底物噬菌粒。通过酶切、DNA测序和核酶反应验证了该方法的准确性。通过DNA测序，我们观察到100%的白色菌落含有单一寡核苷酸序列。
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.