Kuijper J L, Wiren K M, Mathies L D, Gray C L, Hagen F S
ZymoGenetics, Inc., Seattle, WA 98105.
Gene. 1992 Mar 15;112(2):147-55. doi: 10.1016/0378-1119(92)90370-5.
This paper describes the construction of 'Prime' cloning vectors, which include phage lambda and plasmid vectors useful for functional cloning in oocytes, yeast, and mammalian cells, and their use in a 'Prime' cloning system. The system takes advantage of the very active and precise 3' exonuclease activity of T4 DNA polymerase to produce single-stranded (ss) ends (cut-back) of vector and insert DNA. This results in the highly efficient directional cloning of cDNA and PCR-amplified DNA. The system obviates the need to digest insert DNA with a restriction endonuclease to unveil cloning sites, and thus eliminates the chance of internal digestion of the insert DNA. The cloning of PCR-amplified DNA, which is sometimes difficult, is made routine with this system. The 'Prime' sequence is included in vector cloning sites and cDNA and PCR primers. The 'Prime' sequence was chosen so that the ss sticky ends are nonpalindromic and will hybridize only to the appropriate partners. This makes cloning with the 'Prime' system very efficient, because neither the vector nor insert DNA is lost to unproductive self-hybridization.
本文描述了“Prime”克隆载体的构建，其中包括可用于卵母细胞、酵母和哺乳动物细胞功能克隆的λ噬菌体和质粒载体，以及它们在“Prime”克隆系统中的应用。该系统利用T4 DNA聚合酶非常活跃且精确的3'核酸外切酶活性来产生载体和插入DNA的单链（ss）末端（回切）。这导致cDNA和PCR扩增DNA的高效定向克隆。该系统无需用限制性内切酶消化插入DNA以暴露克隆位点，从而消除了插入DNA内部消化的可能性。用该系统可使有时较为困难的PCR扩增DNA的克隆变得常规化。“Prime”序列包含在载体克隆位点以及cDNA和PCR引物中。选择“Prime”序列是为了使ss粘性末端是非回文的，并且只会与合适的配对物杂交。这使得使用“Prime”系统进行克隆非常高效，因为载体和插入DNA都不会因无效的自我杂交而损失。
