Msec Polymerase Chain Reaction with fMolar Detection Sensitivity using SPR

使用 SPR 进行具有 fMolar 检测灵敏度的 Msec 聚合酶链反应

基本信息

批准号：
7201611
负责人：
DONALD K ROPER
金额：
$ 7.26万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-03-15 至 2008-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7201611
关键词：
3-Dimensional Adenoviruses Alleles Amines Base Pairing Binding DNA DNA amplification Detection Electrons Exhibits Extinction (Psychology)Fingerprint Fluorescein Fluoresceins Fluorescence Fluorescent Dyes Gene Mutation Genomics Genotype Globin Gold Human Label Lasers Link Measures Metals Methods Monitor Mutagenesis Mutation Numbers Oligonucleotides Phase Photons Physiologic pulse Point Mutation Polymerase Polymerase Chain Reaction Pulse taking Pump Range Rate Reaction Time Refractive Indices Reporting Sample Size Sampling Sapphire Signal Transduction Single Nucleotide Polymorphism Solutions Speed Spottings Surface Surface Plasmon Resonance System Taq Polymerase Temperature Thymine Time Titanium Work absorption base carboxyl group deoxyribonucleoside triphosphate gene cloning high throughput analysis macromolecule millisecond monolayer nanoscale novel size time use

项目摘要

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.

描述（由申请人提供）：聚合酶链反应（PCR）具有许多应用，包括基因分型突变，遗传指纹，基因克隆和诱变。用于PCR的实验室片和微阵列格式可通过微型引物或等位基因特异性引物伸长来对突变和单核苷酸多态性进行高吞吐量分析。但是，PCR的速度，灵敏度和样本大小受荧光标签的热响应时间缓慢和吸收横截面低的限制，尤其是在高通量格式的情况下。我们提出了对表面等离子体共振（SPR）的新颖使用，以同时诱导DNA样品中的热耗散和共振吸收性检测，以提高DMA扩增速率和实时检测敏感性，同时降低所需的样品体积。 SPR是被异常谐振光子极化的DELEACALIGED贵金属电子的集体振荡。它产生的吸收横截面比荧光染料高106倍，从而使DNA的无标签检测低至1-500 femtomoles（Goodrich等，2004），点突变选择性因子约为105：1（Park等，2002）。但是，尚未报告SPR诱导的DNA扩增。 SPR电子振荡还可以在20-200 nm的金色表面范围内消散Picseconds中的热能，这将允许在女权粉的样本量中每秒1667碱基对（BP）的DNA伸长率。我们假设SPR诱导的DNA扩增可以在毫秒内完成30'PCR循环，以鉴定在女性体材料样品中扩展的替代型。该提案的具体目的是：（1）在PCR中合成SPR活性金（AU）表面，该表面在500至700 nm的波长下表现出最佳的共振吸收和热耗散； (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使用taq和phusion（TM）聚合酶的P-Globin使用正向/反向引物对PC03/PC04和RS42/KM29对，分别通过从deoxyribibribribe deoxyribibe中的局部质量变化（DNA）伸长率（4）。分别与23 bp探针的杂交。我们假设开发这些方法用于SPR诱导的DNA扩增和检测将成为在实验室芯片和微阵列格式中高度平行的PCR的基础，从而成为基因型特异性突变，包括单核苷酸多态性。