Msec Polymerase Chain Reaction with fMolar Detection Sensitivity using SPR

描述（由申请人提供）：聚合酶链反应（PCR）具有许多应用，包括基因分型特定突变、遗传指纹、基因克隆和诱变。用于PCR的芯片实验室和微阵列形式通过微测序或等位基因特异性引物延伸提供突变和单核苷酸多态性的高通量分析。但是PCR的速度、灵敏度和样本量受到缓慢的热响应时间和荧光标记的低吸收截面的限制，特别是在高通量格式中。我们提出了一种新的使用表面等离子体共振（SPR），同时诱导DNA样品中的热耗散和共振吸收检测，以增加DMA扩增速率和实时检测灵敏度，同时减少所需的样品体积。SPR是由入射共振光子极化的离域贵金属电子的集体振荡。它产生的吸收截面比荧光染料高>106倍，允许低至1-500飞摩尔的DNA的无标记检测（古德里奇等人，2004），点突变选择性因子约为105：1（Park等人，2002年）。然而，SPR诱导的DNA扩增尚未报道。SPR电子振荡还在金表面的20-200 nm内以皮秒耗散热能，这将允许在毫微微升的样品尺寸中每秒1667个碱基对（bp）的DNA延伸速率。我们假设SPR诱导的DNA扩增可以在毫秒内完成30个“PCR”循环，以鉴定一个毫微微升样品中的毫微微摩尔扩增子。（1）合成用于PCR的SPR活性金（Au）表面，其在500至700 nm的波长范围内表现出最佳的共振吸收和热耗散;（2）将两个正向/反向引物对和两个聚合酶分别固定在烷硫醇修饰的Au表面上，并表征它们与（3）利用SPR从人基因组DNA模板中扩增出珠蛋白片段，并利用荧光共振增强转移（FRET）扩增出珠蛋白片段的效率;（3）通过SPR诱导热循环，使用正向/反向聚合酶，用Taq和Phusion（TM）聚合酶从人基因组DNA模板中扩增110-和536-bp的β-球蛋白片段反向引物对分别为PC 03/PC 04和RS 42/KM 29;和（4）通过监测分别来自脱氧核苷三磷酸（dNTP）添加和来自扩增子与23-bp探针杂交的局部折射率的无标记变化，真实的实时检测DNA延伸和扩增。我们假设开发这些用于SPR诱导的DNA扩增和检测的方法将成为以芯片实验室和微阵列形式在纳米级水平高度平行PCR的基础，以基因型特异性突变包括单核苷酸多态性。

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.