1. Introduction
Solid-phase amplification (SP-PCR) is a technique that immobilizes one or two primers on a solid support, allowing DNA amplification on the surface. SP-PCR offers significant advantages over liquid-phase amplification, such as the ability to detect trace samples [
1] and the immobilization of products on the surface, facilitating the detection of results. In addition, the immobilization of primers significantly minimizes false positive signals caused by primer dimerization [
2,
3,
4]. These advantages give SP-PCR great application prospects in the fields of high-throughput gene sequencing [
5], single nucleotide polymorphism (SNP) [
6], and multiplex diagnosis [
7].
Traditional SP-PCR typically relies on high-temperature denaturation, which leads to a decrease in surface DNA density [
8,
9], reducing the efficiency of SP-PCR. Over the years, researchers have immobilized DNA on the solid support through covalent coupling. However, even with the use of various immobilization methods such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), benzene-1,3,5-triacetic acid (BTA), and various sulfonated analogues, the problem of heat-induced DNA loss after thermal cycling remains common. These methods lead to a loss of 40–60% of DNA after multiple thermal cycles [
5,
10], and even significant DNA loss can be observed in a short period of time [
9,
11]. Faced with this challenge, our research aims to study other gentler methods to lower the overall temperature of the reaction and improve the issue of DNA loss in SP-PCR, thereby enhancing the efficiency and accuracy of SP-PCR.
Formamide (FA) is a chemical reagent that denatures the secondary structure of DNA by generating hydrogen bond coupling with the DNA bases, thus lowering the melting temperature (Tm) of DNA [
12,
13]. Therefore, it is often used for DNA denaturation in various hybridization techniques, such as fluorescence in situ hybridization [
14,
15] and blot hybridization [
16]. The conventional denaturation temperatures of PCR are typically between 94 °C and 98 °C, but FA can reduce them to 80 °C or even lower due to its hydrogen bond-breaking properties [
17]. The fact that DNA can be amplified at comparatively lower temperatures suggests a reduced risk of heat-induced DNA loss and degradation. This may have a positive effect on SP-PCR, particularly given that the immobilized primers exhibit greater stability in a milder environment. This enhanced stability can significantly contribute to improving the efficiency of the amplification process. Currently, research mainly focuses on how FA affects DNA stability in liquid-phase environments, while there are relatively few studies on its effects on a DNA microarray, and these studies have several limitations [
18,
19]. Specifically, these studies only explored the effects of FA at concentrations below 20% and 45%, and mostly employed shorter oligonucleotides as samples. Furthermore, they did not fully account for the potential impact of solid surface effects on immobilized DNA. Given that the extent of FA’s effect on the Tm is closely related to the (G+C) composition and helical structure of DNA [
13], these limitations mean that their conclusions may not apply to other various experimental platforms, particularly when it comes to long strands of DNA with complex (G+C) compositions and solid surfaces with different chemical properties. Hence, a thorough and systematic study is still needed to solidify the theoretical foundation for real-world use and enhance comprehension of FA’s influence on immobilized DNA on the surfaces within the specified experimental settings.
This study explored the promoting effects of FA in SP-PCR. Primers were immobilized on the amino surface of the microfluidic chip using zero-length crosslinker 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC)/1-methylimidazole chemistry. Then, DNA microarrays were generated through bridge amplification on the automated device, utilizing both FA denaturation and high-temperature denaturation. We conducted a series of gradient experiments to optimize the denaturation parameters and hybridization conditions of FA. By comparing the results with those obtained from the high-temperature group, we explored the promoting effects of FA on surface hybridization. Furthermore, we compared the thermal stability of immobilized DNA under optimized conditions for both methods and discussed the significance of FA’s role in preserving DNA stability. This study is expected to provide some valuable insights for improving DNA stability and amplification efficiency in SP-PCR.
2. Materials and Methods
2.1. Reagents and Instruments
These included 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 1-methylimidazole (Im) (Macklin Biochemical, Shanghai, China); 20× SSC buffer (GenClone Biotechnology, Beijing, China); 2× Phanta Max Master Mix, VAHTS Universal DNA Library Prep Kit for Illumina V3/ND607, VAHTS HiFi Amplification Mix/N616, VAHTS DNA Adapters for Illumina/N805, VAHTS DNA Clean Beads, Taq DNA polymerase, dNTP Mix (Vazyme Biotech, Nanjing, China); 10,000× SYBR Green I, Betaine and Bovine Serum Albumin (Solarbio, Beijing, China); formamide (Yuanye, Shanghai, China); OP AminoSlideTM (CapitalBio Technology, Chengdu, China); Qubit 1X dsDNA HS Kit, Qubit™ 4 Fluorometer, ProFlex™ 96-Well PCR System (Thermo Fisher Scientific, Waltham, MA, USA); and Agilent 2100 Bioanalyzer System (Agilent, Santa Clara, CA, USA).
2.2. Reaction Device
The device utilized in this study comprises two principal components: a thermal cycling module and a microfluidic module, as depicted in
Figure 1A. The thermal cycling module mainly consists of a Peltier-based semiconductor heating module, in addition to a temperature control panel, a temperature sensor, a radiator, and a copper block. On the other hand, the microfluidic module is made up of three parts: reagent storage chambers, a rotary valve, and an injection pump. The rotary valve alternates among multiple valves to pull the required reagents into the channel for reaction, while the injection pump generates negative pressure to drive the system. Using LabView 2017, this device regulates the fluid route and temperature. The experiment utilizing this device for primer immobilization and amplification was depicted in a flowchart, as shown in
Figure 1B.
2.3. Synthetic Oligonucleotides
In this study, immobilized primers were designed based on the sequence of the adapters used for library construction, ensuring complementarity with a portion of the library sequence during bridge amplification. The primers were designed as follows:
To minimize the steric hindrance on the solid surface, we have added a 10-T sequence to the 5′ position. The 5′ ends of all primers were modified with phosphate groups (5′-P) to form the phosphoramidate bonds with amino groups on the solid surface. The primers P1 and P2, with their 3′ ends remaining unmodified, functioned as forward and reverse primers for SP-PCR. In order to assess the stability of immobilized primers, fluorescent groups (3′-Cy3) were added to the 3′ ends of primers P3 and P4. They were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China).
2.4. Library Preparation
The samples were deep-sea microorganisms provided by the Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, and their genomes were extracted by our colleagues. The V3–V4 region was cloned from the extracted genome using the following PCR mix solution: 2× Phanta Max Master Mix, 0.4 µM forward and reverse primers, and 150 ng DNA. The thermocycling process consisted of the following steps: initial denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 15 s, annealing at 57 °C for 15 s, and extension at 72 °C for 30 s. At 72 °C, a 5 min extension step was then carried out. After that, the amplicons were processed for library preparation using the kits ND607, N616 and N805, and the product was characterized by the Qubit™ 4 Fluorometer and the Agilent 2100 Bioanalyzer.
2.5. Attachment of Oligonucleotides to Aminated Surface
The 5′ ends of primers P1 and P2 are modified with phosphate groups. In the presence of EDC and 1-methylimidazole, these phosphate groups can bind to the amino groups on the chip, forming phosphoramidate bonds. This consequently immobilizes the primers onto the chip. Primers P1 and P2 were prepared as a 1 µM solution in 10 mM 1-methylimidazole containing 40 mM EDC. The solution was pumped into the channel of the microfluidic chip and incubated at 50 °C for 60 min in a humid atmosphere to avoid evaporation. Following incubation, the channel was rinsed with 5× SSC buffer containing 0.1% Tween 20 for 2 min and rinsed and stored in 5× SSC buffer until used.
2.6. Solid-Phase Amplification on the Automated Device
The SP-PCR was first performed based on the method of high-temperature denaturation (high-temperature group). The chip was subjected to a 1 h blocking step with 0.1% BSA containing 5× SSC buffer and 0.1% Tween 20. Subsequently, it was rinsed with 5× SSC buffer and then deionized water sequentially. The PCR mix solution contains 1× PCR Buffer, 0.25 mM dNTPs, 2.5 mM MgCl2, 1 M betaine, 0.4 mg/mL BSA, 0.1% Tween 20, and 0.05 U/µL Taq DNA polymerase. The thermocycling was carried out as follows: denaturation at 95 °C for 1 min, annealing at 45 °C for 2 min and extension at 73 °C for 2 min. Fresh PCR mix was pumped in during the final 20 s of each cycle. This entire procedure was repeated for 40 cycles. At the end of the reaction, the chip was rinsed with 5× SSC buffer containing 0.1% Tween 20, 5× SSC buffer and water sequentially for 3 min each time.
The thermocycling for SP-PCR based on FA denaturation (FA group) was carried out as follows: FA was introduced into the channel, followed by denaturation at 80 °C for 1 min. Subsequently, fresh PCR mix was pumped in, and then annealing was performed at 35 °C for 2 min, followed by extension at 73 °C for 2 min. The whole procedure was repeated for 40 cycles. All other treatments were the same as those in the high-temperature group.
2.7. Visualization of Clusters and Data Analysis
Amplified DNA clusters were stained with a 1× SYBR Green I solution for 5 min. Images of the clusters were captured by a self-made fluorescent microscope, and the schematic diagram of the imaging process is shown in
Figure 2. Finally, the density of the clusters was statistically analyzed by ImageJ (version 1.8.0).
2.8. Evaluation of the Thermal Stability of the Immobilized Primers
Primers P3 and P4 were immobilized on the chip, and thermocycling reactions based on high-temperature denaturation and FA denaturation were performed, respectively, according to the steps mentioned above. Each treatment was repeated multiple times independently. Following the reaction, the same course of treatment was taken. The results were characterized using our fluorescence microscope. Lastly, ImageJ was used to analyze fluorescence intensity on the chips, followed by calculating the mean intensity, and standard deviation. Additionally, the Wilcoxon rank-sum test was performed to assess the statistical significance of the difference between two treatments.
4. Conclusions
In summary, we have successfully achieved the automation of bridge amplification on our device and microfluidic chip, generating DNA microarrays. Firstly, we confirmed that effective SP-PCR can be performed on our thermal cycling/microfluidic device, which is based on the primer’s immobilization through EDC crosslinking chemistry. The clusters exhibited a uniform distribution, with diameters ranging from 2.5 to 4 µm and a signal-to-noise ratio of 2 to 5. This demonstrates the feasibility of our primer’s immobilization strategy, reaction settings, and the potential for automating SP-PCR on microfluidic chips using our reaction and detection device. Based on this experimental foundation, we optimized the use of formamide denaturation, replacing high-temperature denaturation. We determined that 80 °C was the optimal denaturation temperature for formamide. With a final concentration of 6 pM DNA library (diluting with 50% formamide) and annealing at 35 °C, we were able to obtain a maximum cluster density of 2.83 × 104 colonies/mm2. Formamide denaturation exhibited higher sensitivity in SP-PCR, requiring a lower template concentration, operating at a more moderate temperature, and producing a higher cluster density than high-temperature denaturation. This demonstrated the advantageous effect of formamide on surface hybridization. Under optimized conditions, the immobilized DNA loss was 45% in formamide denaturation and 60% in high-temperature denaturation, suggesting that formamide’s capacity to lower temperature improves immobilized DNA stability more. Our results imply that formamide treatment helps preserve DNA stability on solid surfaces and improves the amplification efficiency in SP-PCR. Our knowledge of the formamide mechanism in SP-PCR has been enhanced by this work, which also offers valuable insights for enhancing immobilized DNA stability and amplification efficiency in real-world applications.