There are many different methods and protocols on making your PCR run more efficiently. I recently came across an interesting PCR method called “nanoparticle” PCR. This method seems to attract a lot of attention, because it enhances a PCR by a few orders of magnitude. More interestingly, while the enhancement effect has been reported in a number of different articles, there are a number of theories on the mechanism behind it. So I will walk you through the technology and talk about a few possibilities behind it.
What Is Nanoparticle PCR?
A nanoparticle PCR incorporates small molecular substances with particular physical properties to enhance its reaction. Scientists knew a long time ago that single-stranded binding proteins and DMSO enhanced PCRs. But there are a lot of nanoparticles that are able to enhance the specificity of a PCR. These include quantum dots, single-walled nanotubes, and multi-walled nanotubes—to name a few.1
Liu’s group was the first to show that adding gold nanoparticles at nanomolar concentrations (0.7-13 nM) increased the sensitivity of a PCR from 10 to 1000 times, depending on the PCR system.2 They reasoned that gold nanoparticles have great thermal transfer properties and can, therefore, help ramp up and down a PCR. Similarly, we now use PCR tube/plates made of the thinnest material with high thermal transfer efficiency and the smallest reaction mix for the exact same reason. In addition, better heat transfer decreases PCR times, as shown by photonic PCR, which utilizes light directed at a thin wafer of gold sheet. This system is able to cycle through 50 PCR cycles in 5 min!
Below are two examples of using nanoparticle PCR to enhance sensitivity:
The Cui group successfully used a nanoparticle PCR kit to differentiate between wild-type and gene-deleted (vaccine strain) pseudorabies virus. They reported a 100–1000 fold sensitivity over conventional PCR.3
Cheng’s group used nanoparticle PCR to detect mink enteritis virus from wild, captured mink and reported a 100 fold increase in sensitivity.4
It’s clear that it works, but you must read on to learn the different theories on how nanoparticle PCR actually works.
How Does Nanoparticle PCR Work?
I got interested in writing about nanoparticle PCR, because I wanted to understand how it works. It turns out that there is more than one theory on how it enhances PCR efficiency. So, here are a few explanations about how gold nanoparticles, or nanoparticles in general, increase the sensitivity of a PCR.
Below are the major theories:
- Polymerase adsorption: This proposed mechanism has been supported by many different groups.1, 5–7 Essentially, the gold nanoparticle adsorbs a some of the polymerase and modulates the amount of polymerase available in the system, which might be important in enhancing the specificity of the reaction.
- Primer adsorption: nanoparticles adsorb primer pairs and lowers the melting temperature at duplex formation between perfectly paired and mis-paired primers, which leads to an increase in the specificity of the reaction.1,6
- Product adsorption: the nanoparticles associate with the PCR products and promote efficient dissociation during the denaturing step.1,6
- Greater thermo-conductivity: one study uses primer competition and heat-denatured salmon sperm DNA as an extra competition source and suggests that gold nanoparticles might increase PCR efficiency through its superior thermo conductivity.2,8
- Preference for short PCR products: A slightly different twist says that gold nanoparticles do not increase the specificity of the overall PCR, but rather suppress the formation of long products and favor short short products.9
Increase PCR Sensitivity Through Nanoparticles
So, nanoparticle PCR is another option you want to consider if you want to increase your PCR specificity and efficiency. Which mechanism do you think is working in nanoparticle PCR??? My suspicion is that it is a combination of factors. Please let me know what you think!
- Shen C and Z Zhang. (2013) An Overview of Nanoparticle-Assisted Polymerase Chain Reaction Technology. In Bio-Nanotechnology, edited by Debasis Bagchi CNS,IChE CN, nashi Bagchi FACN, Hiroyoshi Moriyama FACN, and Fereidoon Shahidi FRSC FACS, FAOCS, FCIC, FCIFST, FIAFoST, FIFT, 97–106. Blackwell Publishing Ltd., 201
- Li M, Y Lin, C Wu, and H Liu. (2005) Enhancing the Efficiency of a PCR Using Gold Nanoparticles. Nucleic Acids Research. 33: e184. doi:10.1093/nar/gni183.
- Ma X, et.al. (2013) A Nanoparticle-Assisted PCR Assay to Improve the Sensitivity for Rapid Detection and Differentiation of Wild-Type Pseudorabies Virus and Gene-Deleted Vaccine Strains. Journal of Virological Methods. 193: 374–78. doi:10.1016/j.jviromet.2013.07.018.
- Wang J, et.al. (2015) Development of a Nanoparticle-Assisted PCR (nanoPCR) Assay for Detection of Mink Enteritis Virus (MEV) and Genetic Characterization of the NS1 Gene in Four Chinese MEV Strains. BMC Veterinary Research. 11: 1. doi:10.1186/s12917-014-0312-6.
- Mi L, et.al. (2007) Mechanism of the Interaction between Au Nanoparticles and Polymerase in Nanoparticle PCR. Chinese Science Bulletin. 52: 2345–49. doi:10.1007/s11434-007-0327-5.
- Lou X and Y Zhang. (2013) Mechanism Studies on NanoPCR and Applications of Gold Nanoparticles in Genetic Analysis. ACS Applied Materials & Interfaces. 5: 6276–84. doi:10.1021/am4013209.
- Bai Y, et. al. (2015) Nanoparticles Affect PCR Primarily via Surface Interactions with PCR Components: Using Amino-Modified Silica-Coated Magnetic Nanoparticles as a Main Model. ACS Applied Materials & Interfaces. 7: 13142–53. doi:10.1021/am508842v.
- Lin Y, et.al. (2013) Mechanism of Gold Nanoparticle Induced Simultaneously Increased PCR Efficiency and Specificity. Chinese Science Bulletin. 58: 4593–601. doi:10.1007/s11434-013-6080-z.
- Vu BV, D Litvinov, and RC Willson. (2008) Gold Nanoparticle Effects in Polymerase Chain Reaction: Favoring of Smaller Products by Polymerase Adsorption. ResearchGate. 80: 5462–67. doi:10.1021/ac8000258.