An extension-quenching-extension sequencing on a microarray.
Inherent problems exist with sequencing-by-synthesis (SBS) methods which use fluorescein-labeled nucleotide incorporation into a target template based on a polymerase chain reaction (PCR). These problems include lowering the cost of sequencing and the removal of fluorescence in DNA sequencing for further reading. How can these sequencing problems be resolved? We present a sequencing strategy which we call an extension-quenching-extension sequencing on a microarray based on a two-primer hyperbranched rolling circle amplification (HRCA). The microarray is a high throughput and low-cost tool. The template on a microarray for SBS was prepared by HRCA. The Cy 5-labeled deoxyribonucleoside triphosphate (dNTP) species were incorporated in the extension reactions. We discovered that copper (CuSO(4)) can quench the fluorescence in DNA sequencing because it exhibits an energy-transfer mechanism of quenching from the fluorescein to the bound Cu(2+) ion. The fluorescein label needs to be destroyed after the readout by CuSO(4) before further reading is possible. This paper describes the process used which discovered a successful combination of temperature, concentration, and duration of use for CuSO(4) which successfully quenched the fluorescence. In this experiment, we used a known sequence as a template in order to provide a strategy for sequencing.
Gao
Li
L
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China. oga2001@163.com
Lu
Hua
H
Zhao
Hong
H
Lu
Zuhong
Z
D000818:Animals; D001483:Base Sequence; D019327:Copper Sulfate / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D019793:Fluorescein / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D003854:Deoxyribonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D019327:Copper Sulfate; D019793:Fluorescein