Simin Sharifi 1, Mohammad Samiei 2, Sepideh Zununi Vahed 3, Senem Sunar4, Solmaz Maleki Dizaj 1,5,*
1 Dental and Periodontal Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2 Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran
3 Kidney Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
4 Department of Nanotechnology and Advanced Materials, Mersin University, Mersin, Turkey
5 Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
Solmaz Maleki Dizaj, Dental and Periodontal Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Tel: +98 (41) 33353162, E-mail: firstname.lastname@example.org
Principle, mechanism and advantages of NanoPCR
One of the useful and popular molecular methods is polymerase chain reaction (PCR). However, PCR method was still recurrently impaired by its poor sensitivity, low specificity, false positive results, etc. To improve both productivity and quality of PCR, nanomaterials such as carbon nanomaterials, metal nanoparticles, quantum dots and nano metal oxide have been added into PCR reaction. Nanoparticle assisted PCR (NanoPCR) has received great attention because of its exceptional selectivity, sensitivity and efficiency. In this review article, it was summarized the mechanisms and advantages of the mostly utilized nanoparticles in NanoPCR, such as carbon nanotube, gold, quantum dots, graphene, metallic oxide and composite nanomaterial.
Polymerase chain reaction (PCR) is method of DNA amplification in vitro . PCR technology can be utilized in sequencing of DNA, detection of microorganisms, identification of inherited disorders, expression level and analysis of gene, cloning, gene chip, forensic science and several other useful applications [2-8].
Large quantities of DNA fragments can be obtained due to the exponential amplification of PCR method for genetic analysis and DNA sequencing. One of the drawbacks of PCR method is its low sensitivity, specificity and false negative results, particularly in GC rich sites. Therefore, it is essential to advance novel technologies to improve the efficiency and specificity of PCR amplification. Several PCR enhancers materials have been used such as dimethyl sulfoxide (DMSO), formamide and betaine, glycerol, etc [6, 9, 10]. Although these approaches can improve the efficiency and specificity of PCR reaction, still there are some limitations.
Recently, exceptional physical and chemical properties of nanoparticles such as small size and large surface area cause to they have attracted attention and progressively penetrate into the life sciences field, which significantly promoted the progress of life science and technology [11-13]. Furthermore, the nanoparticles assisted PCR method (NanoPCR) based on the nanoparticles such as carbon nanotubes, quantum dots, gold nanoparticles (AuNPs has been established to considerably accelerate PCR reaction procedure, and improve sensitivity, specificity, efficiency of PCR amplification. In this review article, it was mainly summarized the mechanism and application of PCR amplification technology based on various nanoparticles [14-16].
Effect of nanoparticles in NanoPCR
Nanoparticles have several exceptional properties, such as good surface effect, quantum effect, etc. Nevertheless, each nanoparticle has different and distinctive characteristics in biocompatibility, water-solublility and heat stability, hence, the properties of different nanoparticles on the PCR reaction are diverse . Optimum concentration in reaction, advantages and mechanism of utilized nanoparticle in PCR system summarized in Table. 1.
Table. 1. Advantages, mechanism and optimum concentration of utilized nanoparticle in PCR system
|Assisted nanoparticles||Optimum concentration in reaction||
-Extensive the annealing temperature.
-Improve the specificity of PCR
-Shorten the reaction time and increase the efficiency of amplification.
-Increased Sensitivity by 5‒10 times for conventional PCR , and 104 times for quantitative PCR
-Good thermal conductivity
-Increase the specificity with contribute to the small fragments amplification.
-could shorten the reaction time and improve its efficiency and specificity.
–AuNPs had comparable performance as single-stranded binding protein (SSB), therefore, The selective binding of AuNPs with ssDNA can inhibit the mismatch of templates and primers, and thus decrease the of non-specific amplification and mismatching likelihood of the reaction.
-AuNPs could dynamically regulate activity of polymerase and played a role alike HS-PCR (hot-start PCR), which efficiently hindered non-specific amplifcation.
– At lower temperatures, AuNPs decreased the activity of high fidelity Pfu polymerase.
-AuNPs improve the thermal conductivity and efficiency of thermal cycling, thus improving the efficiency and specificity.
-AuNPs promotes melting of DNA template, significantly improves the dissociation rate of primers, templates, and shortens the reaction time.
|Quantum dots||–||-Improve the specificity of reaction, particularly for short fragments of DNA.
– Strong specific amplification effect in the annealing temperature is in range of 30‒45 °C.
– Reduce non-specific amplification in multiplex PCR, thus considerably improving the specificity of its amplification.
– Decrease the amplification time, and increase the amplification rate.
– Low cost, repeatability universality and can be used to high-throughput amplification of a large number of actual samples.
|-Improving effect of QDs is because of the nucleus itself and the polymerase, rather than its surface state.
-Interaction between QDs and nonspecific adsorption of Taq polymerase was not as strong as that of high-fidelity polymerase, so there was no noticeable effect on normal Taq polymerase.
-QDs played a good hot start effect
-In common PCR, QDs could dynamically regulate the activity of high fidelity polymerase (Pfu), and realized in vitro the efficient DNA replication procedure alike to “hot start”
-QDs had alike thermal initiation effects
(MWCNTs) : 0.8‒1.6 mg
|-single-walled carbon nanotubes (SWNTS) could improve PCR amplification
-A suspension of carbon nanopowder (CNP) could considerably improve the amplification productivity of long fragment PCR and repeated PCR.
|-SWNTS can ensure the DNA polymerase activity even without Mg2+
|Graphene||12–60 mg mL-1||-Good heat conduction and combination with single stranded DNA.
-increased specificity of PCR
-Increase the specificity and selectivity of PCR.
|-Exceptional thermal conductivity of GNFs is the main cause for improving the efficiency of PCR.|||
|TiO2||0.4 nM||-TiO2 (0.2 nM) could be utilized to
amplify the very low template concentration of target DNA.-PCR yield is three times or higher than that with large diameter (25 nm).
|-Great heat transfer effect of TiO2 accelerates the DNA denaturation, thus shortening the overall reaction time.|||
|8–10 nM||-Great magnetization and superparamagnetism, and their surfaces were more functional related to other nanoparticles.||Fe3O4 nanocomposites modified with carboxyl groups could increase the sensitivity of PCR.|||
|MWCNTs and PDMS||-increase the amplification efficiency
-reaction time was shortened
-the efficiency of biological process could be controlled more precisely
|-Because of their good thermal conductivity and thermal stability, increase the amplification efficiency of PCR.
– By changing the content of composite carbon nanotubes, the efficiency of biological process could be controlled more precisely.
|PDA silica and
C-PDA silica nanoparticles
|-The composite nanomaterials could improve the environment of the PCR system, making it more conducive to the reaction.||-Surface of C-PDA particles could provide multiple contact sites for binding polymerases and primers, which improved the stability of the system. Furthermore, the carbonized poly dopamine-coated SiO2 had the most noticeable effect.|||
General Mechanism of nanomaterial in NanoPCR
Nonspecific amplification and primer mismatch extremely affect the specificity and sensitivity of PCR reaction. Nanoparticles can significantly increase the sensitivity, specificity and yield of PCR because of their heat conduction, great surface properties and specific attaching to single-stranded DNA or proteins. The nanoparticles mechanism for PCR optimization is mostly based on the two aspects, which are thermal conductivity and surface effects of nanoparticles.
Surface properties of nanoparticles can effect on PCR with the interaction with polymerase, and effect on the primer or template DNA [15, 30].
Nanoparticles could change the concentration of the polymerase via binding with the polymerase enzyme by good surface properties. It was explored the effect of nanomaterials on the mismatch system and addition of nanomaterials would result in a lower temperature of the mismatch process. The attaching of nanomaterials to product of PCR would promote the decomposition upon denaturation. Additionally, low concentration of nanoparticles would prevent the amplification of long fragments, but high concentrations of nanoparticles would prevent the amplification of small fragments. Nanomaterials did not prevent the non-specific amplification because of mismatch. The surface characteristics of nanoparticles have a considerable effect on PCR reaction, so understanding of surface properties of each nanomaterials is a great helps for nanoparticles optimizing for PCR reaction. The good thermal conductivity of nanomaterials also plays a critical role in temperature changes in the cycles of PCR system. Nanomaterials have good heat conduction performance, which improve the ascending/cooling rate of PCR, thus improving of the system efficiency and shortening the reaction time [16, 31, 32].
Conclusion and future prospect
Studies provided strong foundations for investigating the role of nanoparticles in improving the PCR efficiency, its application in different PCR systems including real-time PCR, and the interaction of nanoparticles with the biological system. In future investigations, combination of nanomaterial-based enhancer and even combination of different enzymes should be exanimated in a diversity of PCR reaction to analyze their possible effects. Furthermore, new nanoparticles are being explored, and surface modifications of these nanoparticles are also considered to further improve the PCR efficiency. These nanoparticles should also be tested for their probable catalytic properties in other enzymatic reaction. Additional surveys are needed to study whether the nanoparticles have any effect on the DNA polymerase and structural characteristic of DNA template during the PCR reaction.
The authors specify that there are no financial supports
for this research.
Conflict of interests
The authors have no conflict of interest.