A strategy for sequencing based on rolling-circle amplification on a microarray.

The sequencing of DNA fragments has become an important facet of the study of genomes. Parallel genomic DNA fragments displayed on a microarray play a key role in producing a template in next generation DNA sequencing. Here, we developed a technique to display parallel genomic DNA fragments based on the reaction with Bst DNA polymerase on a microarray. One of the hyperbranched rolling circle amplification (HRCA) primers was modified with acrylamide. HRCA products could be localized near the respective templates in polyacrylamide gel. DNA colonies produced by HRCA were displayed massively and in parallel on a microarray. Then the probe labeled with Cy 5 was hybridized to DNA colonies and extension reactions with Cy 5-labeled deoxyribonucleoside triphosphates (dNTPs) were carried out. The results show the signals of hybridization and extension reactions could be obtained. This provided a strategy for high-throughput sequencing.