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）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。实验室芯片和微阵列格式的PCR提供高通量分析突变和单核苷酸多态性的微测序或等位基因特异性引物延伸。但是，PCR的速度、灵敏度和样本量受到慢热响应时间和荧光标记低吸收截面的限制，特别是在高通量格式下。我们提出了一种新的使用表面等离子体共振（SPR）来同时诱导DNA样品中的热耗散和共振吸收检测，以提高DMA扩增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飞摩尔的无标记DNA检测（Goodrich等人，2004），点突变选择性因子为105:1 （Park等人，2002）。然而，spr诱导的DNA扩增尚未见报道。在金表面20-200纳米范围内，SPR电子振荡也以皮秒为单位耗散热能，这将使DNA的延伸率达到每秒1667个碱基对（bp）。我们假设SPR诱导的DNA扩增可以在几毫秒内完成30个“PCR”循环，以识别飞升样品中的扩增子。本课题的具体目标是：(1)在500 ~ 700 nm波长范围内，合成具有spr活性的PCR用金（Au）表面，该表面具有最佳的共振吸收和热耗散；(2)分别将两个正向/反向引物对和两个聚合酶固定在烷硫醇修饰的Au表面上，用SPR分析了它们与人类基因组DNA模板中的3-珠蛋白片段的相互作用，并用荧光共振增强转移（FRET）分析了它们的扩增效率；(3)利用SPR诱导热循环，利用Taq和Phusion（tm）聚合酶分别利用PC03/PC04和RS42/KM29正/反向引物对从人类基因组DNA模板中扩增110和536 bp的p-珠蛋白片段；(4)通过监测脱氧核糖核苷三磷酸（dNTP）添加和扩增子杂交对23-bp探针的局部折射率的无标记变化，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在芯片实验室和微阵列格式的纳米级高度平行PCR的基础，以对包括单核苷酸多态性在内的特定突变进行基因分型。

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.