title: Loop-linker PCR: an advanced PCR technique for genome walking.
abstract: In this article, we developed a novel PCR method, termed loop-linker PCR, to isolate flanking sequences in transgenic crops. The novelty of this approach is its use of a stem-loop structure to design a loop-linker adapter. The adapter is designed to form a nick site when ligated with restricted DNA. This modification not only can prevent the self-ligation of adapters but also promotes the elongation of the 3' end of the loop-linker adapter to generate a stem-loop structure in the ligation products. Moreover, the suppressive effect of the stem-loop structure decreases nonspecific amplification and increases the success rate of the approach; all extension products will suppress exponential amplification except from the ligation product that contains the specific primer binding site. Using this method, 442, 1830, 107, and 512 bp left border flanking sequences were obtained from the transgenic maizes LY038, DAS-59122-7, Event 3272, and the transgenic soybean MON89788, respectively. The experimental results demonstrated that loop-linker PCR is an efficient, reliable, and cost-effective method for identifying flanking sequences in transgenic crops and could be applied for other genome walking applications.
authors.affiliation: Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
authors.forename: Quoclinh
authors.initials: Q
authors.lastname: Trinh
authors.forename: Hui
authors.initials: H
authors.lastname: Shi
authors.forename: Wentao
authors.initials: W
authors.lastname: Xu
authors.forename: Junran
authors.initials: J
authors.lastname: Hao
authors.forename: Yunbo
authors.initials: Y
authors.lastname: Luo
authors.forename: Kunlun
authors.initials: K
authors.lastname: Huang
mesh_terms: D024506:5' Flanking Region; D016386:Chromosome Walking / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018744:DNA, Plant / Q000032:analysis*; D018745:Genome, Plant; D055029:Inverted Repeat Sequences / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013025:Glycine max / Q000235:genetics*; D003313:Zea mays
chemical_list: D017931:DNA Primers; D018744:DNA, Plant; D004262:DNA Restriction Enzymes