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| Label me SERRS-able. Two forms, normal (+) and mutant (Δ), of the cystic fibrosis gene were amplified using PCR primers labeled with HEX (H) and rhodamine (R) and assayed by surface-enhanced resonance Raman scattering (SERRS). (Adapted from Graham, D.; et al. Anal. Chem. 2002, 74 (5), 1069–1074.) |
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Determining whether a specific gene sequence exists in an individual is critical to disease diagnosis and pharmacogenetic profiling. Typically, this is accomplished through methods such as polymerase chain reaction (PCR) amplification of the target sequence using either fluorescently labeled PCR primers or deoxynucleotides. But overlapping absorption and emission spectra from the different fluorophores complicate matters and might introduce lengthy purification steps. Surface-enhanced resonance Raman scattering (SERRS), however, easily differentiates between labels and does not require the use of fluorophores. So Duncan Graham and colleagues at the University of Strathclyde (Glasgow, Scotland) developed a SERRS system to distinguish between normal and mutant versions (alleles) of various genes (Anal. Chem. 2002, 74 (5), 1069-1074).
The researchers amplified a gene that encodes for the class II major histocompatibility antigen, which is involved in the immune response, by using a PCR primer labeled with the fluorophore 2,5,2´,4´, 5´,7´- hexachloro-6-carboxyfluorescein (HEX). Excess primer, which would interfere with the SERRS measurement, was removed from the PCR reactions by binding the newly generated DNA to a probe and then binding the complex to a bead. After the beads were thoroughly washed, the labeled PCR products were released by heating at 95 °C. The SERRS signal indicated the presence of appropriately sized HEX-labeled DNA fragments.
The researchers then tested the system on normal (+) and mutant (Δ) alleles of the cystic fibrosis gene to see if they could distinguish between people who carried two copies of the same allele (+/+ or Δ/Δ) or one of each (+/Δ). They labeled PCR primers with HEX for the normal allele and rhodamine for the mutant allele, and then tested pretyped DNA samples. The assay successfully produced three distinct spectra corresponding to the three possible outcomes: The +/+ sample showed HEX spectra, the Δ/Δ sample showed rhodamine spectra, and the +/Δ sample showed combined HEX–rhodamine spectra. According to the researchers, the method shows distinct advantages over traditional fluorescent labeling and, with further refinement, considerable promise.
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