This research was conducted through bioinformatics and experimental design. Bioinformatics design was used to design primers from the PIP gene sequence from NCBI and analyze the secondary structure from OligoAnalyzer. Experimental design was used to optimize the annealing temperature of the PIP gene primer with the C. annuum genome through gradient PCR and visualized with electrophoresis.

a. Design Primer

The reference sequence measuring 1223 bp with accession number XM_016711608.2 was entered into the search field on NCBI (Yin et al., 2014). Then, primer selection will appear by pressing the pick primer tools. The primer candidates recommended by NCBI primer BLAST will be selected based on primer length of 18–22 bp, GC percentage between 40–60%, lowest 3' self-complementarity, and product length (Prediger et al., 2024; (Sasmito et al., 2014)). Primer specificity was evaluated using Primer-BLAST by aligning the primer sequences against the NCBI database to ensure that each primer pair produced a single target amplicon in Capsicum annuum and showed no significant amplification with non-target species.

b. Secondary Structure Analysis of Primers

Secondary structure analysis includes dimers and hairpins using OligoAnalyzer software by IDT (https://sg.idtdna.com/pages/tools/oligoanalyzer). The sequences of the forward and reverse primers selected from the NCBI primer picker tool were entered into the analysis column on OligoAnalyzer. The dimer structures to be analyzed included self-dimers and heterodimers, as well as the hairpin structures of each primer. Primer secondary structures were considered acceptable when the predicted ΔG values were greater than −9 kcal/mol, indicating weak or unstable secondary structures (Prediger, 2024).

c. RNA Extraction

Total RNA was extracted using Geneaid Total RNA Mini Kit (Plant) from four-day-old germinated chili seeds of Capsicum annuum that were germinated in the Plant Physiology Laboratory, Universitas Andalas, Indonesia. Approximately 50 mg of chili seeds tissue was processed following the manufacturer’s protocol. The samples were frozen in liquid nitrogen and ground into a fine powder, followed by lysis with 500 µL RB buffer supplemented with 5 µL β-mercaptoethanol, incubation at 60 °C for 5 minutes, and filtration using a filter column. The clear filtrate was mixed with absolute ethanol to bind the RNA, then applied to the RB column and centrifuged. To reduce genomic DNA contamination, DNase I + DNAse I RB treatment was performed in-column. This was followed by a washing step using W1 buffer and wash buffer, followed by a column matrix drying step. Pure RNA was eluted using 50 µL and its quantity was checked with a nanospectrophotometer. RNA purity was assessed based on the absorbance ratios, with acceptable ranges of 1.8–2.2 for A260/280 and >1.7 for A260/230 (Wieczorek et al., 2012). Then, a sample with a concentration of 50 ng/µL was obtained after dilution and stored in a freezer at −80 °C.

d. cDNA synthesis

cDNA synthesis was performed using ReverTra Ace™ qPCR RT Master Mix with gDNA Remover according to the kit protocol. Total reaction volume of 8 μL consisting of 4x DN Master mix 2 μL, RNA template 50 ng/μL 2 μL, and nuclease-free water 4 μL. The reaction mixture was gently mixed and placed in a thermocycler. The reverse transcription reaction was carried out at 37 °C for 15 min, followed by 50 °C for 5 min, and enzyme inactivation at 98 °C for 5 min. The synthesized cDNA was then stored at −20 °C until further use.

e. PCR Gradien

Optimization of the primer annealing temperature was performed using PCR gradient analysis. The total volume of the PCR reaction was 25 µL, consisting of 12.5 µL Bioline RedMix PCR, 10.5 µL nuclease-free water, 1 µL cDNA template, and 1 µL each of forward and reverse primers. Each PCR reaction was performed once. The PCR cycling conditions were performed according to Yin et al. (2015), consisting of an initial denaturation at 95°C for 1 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 55–59°C, and extension at 72°C for 30 s, with a final extension at 72°C for 5 min. The amplified products were separated by electrophoresis on a 2% agarose gel prepared in 1× TAE buffer. Fragment sizes were estimated using a 100 bp DNA ladder (Bioline) and visualized using a Uvitec gel documentation system.

RNA extraction from Capsicum annuum seeds was successfully carried out and its quality was analyzed using a Nano spectrophotometer. The RNA concentration obtained was 321.4 ng/µL with an absorbance ratio of A260/280 of 2.1 and A260/230 of 2.2. These values are within the good range for further analysis, namely 1.8–2.2 for the A260/280 ratio and >1.7 for the A260/230 ratio (Wieczorek et al., 2012). According to Gudenschwager et al. (2012), an A260/230 ratio above 1.8 indicates pure total RNA without polyphenol and polysaccharide contamination, while an A260/280 ratio in the range of 1.8–2.2 indicates low protein contamination. Therefore, the results obtained indicate that RNA isolated from C. annuum seeds has good purity and is suitable for further molecular analysis.

Designing primers is highly recommended using the BLAST primer software available on the NCBI website (Ye et al., 2012). Based on NCBI Primer-BLAST, nine primer pairs were recommended for amplifying the PIP gene in C. annuum. Based on Table 1, the forward and reverse primers from pair 1 to pair 9 have a similar size of 20 bp. The GC percentage ranges from 50% to 55%, the melting temperature is 60^oC, and the 3' self-complementarity is between 0 and 3 bp. The product length of pairs 1 to 9 varies from 80 to 286 bp.

Guanine (G) and Cytosine (C) are nucleotide bases that have three hydrogen bonds. Therefore, GC bonds are stronger than Adenine (A) and Thymine (T) bases, which only have two hydrogen bonds (Marliana et al., 2015). This is an important consideration when selecting primers. In primer design, the amount of guanine and cytosine is referred to as the GC percentage (%GC). The GC value should be in the range of 40-60% (Wang, 2016)(Masnaini et al., 2023) with an ideal percentage of 50% (Prediger et al., 2024). If the primer has a low %GC, the efficiency of the primer to bind to the template sequence will decrease (Masnaini et al., 2023). This is related to the Tm of the primer because the number of G and C bases affects the melting temperature of the primer (Sari, 2018). The data in Table 1 shows a %GC of 50–55%, which is within the ideal range for use.

Specific primers are composed of a pair of primers, namely forward and reverse primers (Li et al., 2012). The forward primer directs DNA synthesis in the 5′–3′ orientation, whereas the reverse primer facilitates synthesis from the opposite strand in the 3′–5′ direction (Baker et al., 2016)(Aurora et al., 2025). Primer length is a critical factor influencing binding specificity to the target template sequence. In general, primers with lengths ranging from 18 to 22 base pairs are considered optimal (Sasmito et al., 2014). Primers shorter than this range tend to exhibit reduced specificity and may anneal to non-target sequences, while excessively long primers increase the likelihood of secondary structure formation (Syamsidi et al., 2021). Consequently, primer lengths of 20–22 bp are most commonly applied in PCR and qPCR analyses Hidayah et al., 2025 (Chen et al., 2023)(Syamsidi et al., 2021)(Li et al., 2012). As shown in Table 1, all primer pairs recommended by NCBI Primer-BLAST possess a uniform length of 20 bp.

Another parameter to consider when selecting primers from the pairs listed in Table 1 is the self3' complementarity value. This value appears when there is a base match at the 3' end of the primer that can trigger the formation of primer dimers that can be extended by DNA polymeraseThis factor influences the efficiency of primer binding to the target sequence. Therefore, the self 3' complementarity value has a tolerance limit of no more than 3 bases (Handoyo & Rudiretna, 2000). In Table 1, the self 3' complementarity values that meet the requirements are in pairs 2, 3, 4, 5, 7, and 8. However, pairs 3, 4, 5, and 7 have short amplification product lengths and are not suitable for amplification with qPCR. An amplification product that is too short can cause mispriming, allowing other fragments to be amplified by that fragment (Astari et al., 2021). Holm et al (2021) stated that an amplification product length of 200–400 bp can increase the quantification cycle difference between DNA from living and dead cells. This is important in qPCR to better reflect the actual biological conditions of an activity being observed via qPCR.

The specificity of the selected primer pairs 2 and 8 was examined using Primer-BLAST from NCBI. Based on Figure 1a and Figure 1b, each primer candidate from pairs 2 and 8 is specific to the species C. annuum. There were no significant matches to the genomes of other species. This indicates that the possibility of primers binding and amplifying non-target sequences is very low. (Ye et al., 2012) stated that Primer-BLAST is an effective tool for evaluating and designing primers specific to genomes. Primer specificity is a key factor in the success of PCR amplification, as non-specific primers can produce non-specific products and reduce reaction efficiency (Hafzari et al., 2024). Therefore, pair 2 and pair 8 were selected for in silico secondary analysis by OligoAnalyzer.

The secondary structures in the form of hairpins, self-dimers, and heterodimers of pair 2 and pair 8 were analyzed using OligoAnalyzer software. The results of the secondary structure analysis showed that both primer pairs had relatively low hairpin ΔG values (Table 2). This indicates a tendency toward the formation of weak hairpin structures. However, a significant difference is seen in the potential for self-dimer formation. Pair 8 shows a very negative self-dimer ΔG value of -9.75 kcal/mol, while pair 2 has a higher and more positive self-dimer ΔG value, indicating a less stable secondary structure.

Secondary structure analysis was conducted using OligoAnalyzer to support the development of high-quality primer designs (Caro et al., 2022). This tool enables the assessment of potential secondary structures, including hairpins and primer dimers, by analyzing Gibbs free energy (ΔG) values. The probability of primer annealing to the target sequence and subsequent extension by DNA polymerase is influenced by changes in Gibbs free energy (ΔG), as this parameter reflects the thermodynamic stability of primer interactions and the tendency to form non-specific structures (Meagher et al., 2018). Hairpin structures occur when a primer folds back and binds to itself (Chuang et al., 2013), whereas primer dimers arise from interactions between primers, either as self-dimers or as heterodimers formed between forward and reverse primers (Fakih et al., 2021).

According to Prediger (2024), the ΔG value in hairpins, self-dimers, and heterodimers should be in the weak range, with a threshold of around -9.0 kcal/mol, because a very negative ΔG value reflects a stable secondary structure. (Yang et al., 2020) stated that the ΔG value in the primer must be greater than -6 kcal/mol. Based on these criteria, pair 8 has the potential to form a stable self-dimer, thereby inhibiting amplification efficiency. Conversely, the ΔG value of the self-dimer in pair 2 indicates that the secondary structure formed is unstable or may not form at all. Therefore, pair 2 was selected and named 2_CaPIP, then proceeded to the primer synthesis stage and further experimental testing.

Annealing temperature (Ta) is regarded as a critical factor influencing primer performance and the success of amplification reactions (Fraige et al., 2013). Ta refers to the temperature range at which primers can stably anneal to DNA or RNA templates (Syamsidi et al., 2021). In comparison with melting temperature (Tm), Ta more accurately reflects the actual conditions required for optimal primer–template binding (Bustin & Huggett, 2017). Consequently, experimental optimization of the annealing temperature is essential, since primer design software typically provides only theoretical Tm values (SantaLucia, 2007).

Optimization of the PCR annealing temperature was performed using five temperatures below the melting temperature of primer pair 2 (Table 1), ranging from 55–59 °C, producing an expected amplicon of 268 bp. According to Mubarak et al. (2020), the optimal annealing temperature is typically 5°C below the Tm. Based on the PCR gradient results shown in the electrophoresis profile in Figure 2, all samples exhibited clear, thick, specific bands without dimers. The presence of a single band at all temperatures indicates that the specificity of the primers has been confirmed (Chen et al., 2023). The results of optimizing several temperatures show that the band at 57.3°C is the brightest and thickest compared to other bands. Clear, thick bands without smearing reflect good DNA bands due to maximal PCR amplification (Irmawati et al., 2003). Therefore, the annealing temperature of 57.3°C is the most optimal for the 2_CaPIP primer to amplify C. annuum cDNA.