Construction and experimental application of a highly efficient temperature-selection T-vector.
T-A cloning is the most commonly used molecular cloning technique in which T-vector is the core tool. T-vectors commonly used now are blue-white positive-selection vectors based on mechanism of alpha-complement of beta-galactose of Escherichia coli. Disadvantages of these traditional T-vectors in application include insufficient positive rate and the existence of false-positive clones, which, especially the latter, makes screening of transformant clones via colony PCR necessary. In the present study, a novel T-vector, based on the strategy of inhibiting the growth of negative transformants by means of a lethal gene and innovative design of insertion site, was developed. The innovative design of the insertion site for PCR fragments not only guarantees complete blocking of the expression of the lethal gene when PCR fragments are inserted, but also eliminates the formation of false-positive clones. All (100%) of 20 randomly chosen grown colonies were shown to be positive via colony PCR in four separate experimental T-A clonings of PCR fragments with lengths between 219 and 2100 bp. The novel T-vector developed in the present study makes colony PCR screening unnecessary, because of its extremely high efficiency of negative transformant inhibition and the mechanism for elimination of false-positive clones. Temperature (42 degrees C)-dependent positive selection greatly simplifies the procedure and lowers the cost of T-A cloning. Furthermore, it is an ideal T-vector for constructing libraries of PCR-amplified DNA fragments such as SSH (suppressive subtraction hybridization) libraries because of its zero negative background performance.
Ma
Yue
Y
School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China. mayue6399@126.com
Li
Anxing
A
Wang
Yongcai
Y
Xie
Mingquan
M
Li
Jing
J
Zhang
Sen
S
Wang
Qiuju
Q