An optimal pooling strategy applied to high-throughput screening for rare marker-free transformants.
An optimal pooling system, called Accurate and Fast Target Screening, has been developed for high-throughput identifying the rare marker-free transformants. This system can identify targets between 10- and 100-fold more efficiently than analysis of individual samples. By calculating the efficiency for different proportions of targets and the optimal group size in a worst case scenario, we are able to estimate an upper limit for the number of tests that are required. The application of this system to determine the transgene in an artificially constructed population of transgenic and non-transgenic wheat lines successfully identified the 10 positive samples located randomly with 990 negative samples using only 92 PCR reactions. The same approach was also applied to determine transgene expression by SDS-PAGE of seed proteins. This system gives unambiguous positive or negative results and should facilitate marker-free transformation.
Zhang
Jinrui
J
China-UK HUST-RRes Genetic Engineering and Genomics Joint Laboratory, Huazhong University of Science and Technology (HUST), Wuhan, Hubei 430074, China.
Xiao
Qin
Q
Li
Kexiu
K
Chen
Mingjie
M
Chang
Junli
J
Luo
Liting
L
Li
Yang
Y
Liu
Yong
Y
Shewry
Peter R
PR
He
Guangyuan
G
D000465:Algorithms; D002478:Cells, Cultured; D004274:DNA, Recombinant; D005819:Genetic Markers; D005983:Glutens / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012639:Seeds / Q000737:chemistry / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes; D014908:Triticum / Q000235:genetics*
D004274:DNA, Recombinant; D005819:Genetic Markers; D005983:Glutens; C037302:glutenin