Massively parallel display of genomic DNA fragments by rolling-circle amplification and strand displacement amplification on chip.
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.
Zhao
Hong
H
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China.
Gao
Li
L
Luo
Junfeng
J
Zhou
Dongrui
D
Lu
Zuhong
Z
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
D005456:Fluorescent Dyes; D004247:DNA; D004259:DNA-Directed DNA Polymerase