Phadnis S H, Huang H V, Berg D E
Department of Microbiology and Immunology, Washington University Medical School, Saint Louis, MO 63130.
Proc Natl Acad Sci U S A. 1989 Aug;86(15):5908-12. doi: 10.1073/pnas.86.15.5908.
We constructed a derivative of transposon Tn5 called Tn5supF for insertion mutagenesis and sequencing DNAs cloned in phage lambda. This element carries a supF amber-suppressor tRNA gene. Its insertion into lambda can be selected by plaque formation by using nonsuppressing (sup0) Escherichia coli for amber mutant lambda phage and sup0 dnaB-amber E. coli for nonamber lambda phage. Tn5supF is just 264 base pairs long. It transposes efficiently and inserts quasi-randomly into DNA targets. The unique sequences near its termini can be used as primer binding sites for dideoxynucleotide DNA sequencing, thus permitting the direct sequencing of DNAs cloned in phage lambda without subcloning.
我们构建了转座子Tn5的一个衍生物，称为Tn5supF，用于插入诱变和对克隆在噬菌体λ中的DNA进行测序。该元件携带一个supF琥珀抑制tRNA基因。通过使用对琥珀突变型λ噬菌体无抑制作用（sup0）的大肠杆菌，以及对非琥珀型λ噬菌体使用sup0 dnaB-琥珀型大肠杆菌，利用噬菌斑形成来选择其插入到λ中。Tn5supF仅264个碱基对长。它能高效转座并准随机插入DNA靶点。其末端附近的独特序列可作为双脱氧核苷酸DNA测序的引物结合位点，从而允许直接对克隆在噬菌体λ中的DNA进行测序而无需亚克隆。
