title: Massively parallel display of genomic DNA fragments by rolling-circle amplification and strand displacement amplification on chip.
abstract: Massively parallel genomic DNA fragments display on chip plays a key role in the new generation DNA sequencing. Here, we developed a new technology to display the parallel genomic DNA fragment massively based on two-step reaction with capital EF, Phi29 DNA polymerase. The genomic DNA fragments were firstly amplified by rolling-circle amplification (RCA) reaction in liquid phase, and then amplified further on the chip by the strand displacement of capital EF, Phi29 DNA polymerase. In our experiments, through DNA colonies produced by two-step amplification reaction T7 genomic DNA fragments are displayed massively and parallely on the chip, which has been verified through hybridizing the probe labeled with fluorescence or extension reaction with fluorescent-dNTP. The significant difference of fluourescence signals between background and displayed DNA fragments could be obtained. Our results show that the method has good reproducibility in experiments, which may be hopeful to serve the high-throughput sequencing.
authors.affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Li
authors.initials: L
authors.lastname: Gao
authors.forename: Junfeng
authors.initials: J
authors.lastname: Luo
authors.forename: Dongrui
authors.initials: D
authors.lastname: Zhou
authors.forename: Zuhong
authors.initials: Z
authors.lastname: Lu
mesh_terms: D004247:DNA / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D016678:Genome / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D005456:Fluorescent Dyes; D004247:DNA; D004259:DNA-Directed DNA Polymerase