Msec Polymerase Chain Reaction with fMolar Detection Sensitivity using SPR

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DESCRIPTION (provided by applicant): Polymerase chain reaction (PCR) has a host of applications including genotyping specific mutations, genetic fingerprinting, gene cloning and mutagenesis. Lab-on-chip and micro-array formats for PCR offer high- throughput analysis of mutations and single nucleotide polymorphisms by minisequencing or allele-specific primer elongation. But speed, sensitivity and sample size of PCR are constrained by slow thermal response times and low absorption cross-sections for fluorescent labels, especially in high-throughput formats. We propose a novel use of surface plasmon resonance (SPR) to simultaneously induce thermal dissipation and resonant absorptive detection in DNA samples to increase DMA amplification rates and real-time detection sensitivity while decreasing required sample volume. SPR is collective oscillation of delocalized noble metal electrons polarized by incident resonant photons. It yields absorption cross-sections >106-fold higher than fluorescent dyes, allowing label-free detection of DNA as low as 1-500 femtomoles (Goodrich et al., 2004) with point mutation selectivity factors of ~105:1 (Park et al., 2002). However, SPR-induced DNA amplification has not been reported. SPR electron oscillation also dissipates thermal energy in picoseconds within 20-200 nm of gold surfaces, which would allow DNA elongation rates of 1667 base pairs (bp) per second in sample sizes of femtoliters. We hypothesize SPR- induced DNA amplification could complete 30 'PCR' cycles within milliseconds to identify femtomoles of amplicon in a femtoliter sample. Specific aims of this proposal are: (1) synthesize SPR-active gold (Au) surfaces for PCR which exhibit optimum resonant absorption and thermal dissipation at wavelengths in the range 500 to 700 nm; (2) immobilize two forward/reverse primer pairs and two polymerases separately on alkanethiol-modified Au surfaces and characterize their interaction with (3-globin fragments from human genomic DNA template using SPR and their amplification efficiency using fluorescence resonance enhanced transfer (FRET); (3) induce thermal cycling by SPR to amplify 110- and 536-bp fragments of p-globin from human genomic DNA template with Taq and Phusion(tm) polymerase using forward/reverse primer pairs PC03/PC04 and RS42/KM29, respectively; and (4) detect DNA elongation and amplification in real time by monitoring label-free changes in local refractive-index from deoxyribonucleoside triphosphate (dNTP) addition and from amplicon hybridization to 23-bp probes, respectively. We hypothesize developing these methods for SPR- induced DNA amplification and detection will become the basis for highly parallel PCR at nanoscale levels in lab-on-chip and micro array formats to genotype specific mutations including single nucleotide polymorphisms.