Genotyping single nucleotide polymorphisms using different molecular beacon multiplexed within a suspended core optical fiber

Abstract

We report a novel approach to genotyping single nucleotide polymorphisms (SNPs) using molecular beacons in conjunction with a suspended core optical fiber (SCF). Target DNA sequences corresponding to the wild- or mutant-type have been accurately recognized by immobilizing two different molecular beacons on the core of a SCF. The two molecular beacons differ by one base in the loop-probe and utilize different fluorescent indicators. Single-color fluorescence enhancement was obtained when the immobilized SCFs were filled with a solution containing either wild-type or mutant-type sequence (homozygous sample), while filling the immobilized SCF with solution containing both wild- and mutant-type sequences resulted in dual-color fluorescence enhancement, indicating a heterozygous sample. The genotyping was realized amplification-free and with ultra low-volume for the required DNA solution (nano-liter). This is, to our knowledge, the first genotyping device based on the combination of optical fiber and molecular beacons.

Figure 1.

Schematic diagram of the final stage of the suspended core optical fiber (SCF) core functionalized with dual-type molecular beacons. Picture of the SCF cross section shown on the right side is a scanning electron microscope (SEM) image of the SCF used in this work.

Figure 2.

Experiment setup for fluorescence measurement of the dual-type molecular beacons (MBs) immobilized SCF filled with DNA solutions. SMF and MMF are abbreviations for single mode and multimode mode optical fiber, respectively. The green laser operating at 532 nm was used to excite HEX dyes (for wild-type sequence) and the red laser operating at 638 nm was used to excite Cy5 dyes (for mutant-type sequence).

Figure 3.

Comparison of fluorescence enhancement between dual-type MB immobilized SCF and control SCF upon filling with target DNA solution. The control SCF shows negligible fluorescence enhancement.

Figure 4.

Hybridization test of the dual-type MB immobilized SCF filled with solution containing either one type of DNA sequence, e.g., wild or mutant sequence (homozygous) or both type (heterozygous). Fluorescence enhancement clearly indicates (a,b) the homozygous type or (c) heterozygous type; (d) The averaged value over four measurements of spectra as shown in (a–c), crossing point of HEX and Cy5 fluorescence indicate heterozygousity and well separated values of HEX and Cy5 fluorescence represent homozygousity.