Book of Abstracts: Albany 2007
June 19-23 2007
A DNA Based Single Nucleotide Polymorphism Detection Device
We show that single-stranded branch migration can be used to detect single base mismatches. The detection is done using fluorescence signals. To establish single base mismatches, the Fluorescein-BHQ1 incorporated duplex is placed in the presence of fuel strands with or without a single base mismatch at different positions of a single strand. This experiment has been performed with various base mismatches in the fuel strand to test the efficiency of the technique. The duplex containing Fluorescein on 5?-end of one strand and BHQ-1 on 3?-end of the other having an overhang of 8 bases are placed in the presence of the exact complement of the BHQ-1 containing strand for control. The experiment is performed at room temperature.
The top part of the schematic representation shows the duplex, red strand containing the Fluorescein (Green) dye at 5?-end, while the black strand has Black Hole Quencher-1 (Purple) at its 3?-end and an 8 base overhang at 5?-end. The emission spectrum shows a quenched fluorescence at 517 nm, compared with the donor-only system, using an excitation wavelength of 490 nm. The first step is the addition of the fuel strand that is fully complementary to the black strand. As soon as it is added to the system it binds to the 8 bases overhang region and branch migration starts that will remove the red strand in the second step of the scheme. If the fuel strand is fully complementary it will remove the red strand completely; as a result Fluorescein and BHQ-1 will be separated from each other, increasing the fluorescence intensity. Once the strands are replaced via branch migration to form a base paired duplex with eight extra base pairs, it will show a fluorescence signal, almost comparable to the donor-only system. If there is even a single mismatch present in the fuel strand, then branch migration will stop when it encounters it, maintaining the quenched signal.
This research supported by NIGMS, NSF, ARO and the W.M. Keck Foundation.
Department of Chemistry