[Screening for resistance gene candidate from a genomic TAC library of Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL by pooled PCR].
A pair of degenerate primers were designed based on NBS (nucleotide binding site, NBS) domain of resistance(R) gene and used to perform PCR with cDNA from the translocation line 6VS/6AL of Triticum aestivum-Haynaldia villosa. A clone (N7) characterized with NBS was obtained by sequencing analysis. Two specific primers were designed from the N7 sequence and used to screen a genomic TAC (transformation-competent artificial chromosome, TAC) library of 6VS/6AL consisting of ca. 2 x 10(6) clones. The library was stored as clone pools in twenty-two 96-well plates, each well containing approximately 1000 TAC clones. TAC plasmids were prepared from all the 2112 pools. Using a pooled PCR screening procedure, a positive TAC clone having a 40 kb insert was obtained. The positive clone was confirmed by Southern hybridization with the NBS fragment as a probe. The results indicate that the pooled PCR method is effective for screening of genomic libraries having large number of clones.
Qin
Gen-Ji
GJ
Cytogenetics Institute, Nanjing Agricultural University, Nanjing 210095, China.
Chen
Pei-Du
PD
Liu
Yao-Guang
YG
Fang
Yu-Da
YD
Liu
Da-Jun
DJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D022201:Chromosomes, Artificial; D015698:Genomic Library; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014178:Translocation, Genetic; D014908:Triticum / Q000235:genetics*