title: Construction of tandem repeats of DNA fragments by a polymerase chain reaction-based method.
abstract: We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71 bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.
authors.affiliation: College of Horticulture and Gardening, Hunan Agricultural University, Changsha, People's Republic of China.
authors.forename: Ji-Ren
authors.initials: JR
authors.lastname: Chen
authors.forename: Zi-Niu
authors.initials: ZN
authors.lastname: Deng
authors.forename: Yan-Bin
authors.initials: YB
authors.lastname: Chen
authors.forename: Bo-Wen
authors.initials: BW
authors.lastname: Hu
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Lü
authors.forename: Yue-Lin
authors.initials: YL
authors.lastname: Long
authors.forename: Xing-Yao
authors.initials: XY
authors.lastname: Xiong
mesh_terms: D029681:Arabidopsis Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000138:chemical synthesis*; D017931:DNA Primers / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences / Q000235:genetics*; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D017931:DNA Primers; D004247:DNA