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. 2023 Jan 23:2023:8296847.
doi: 10.1155/2023/8296847. eCollection 2023.

Colorimetry-Based Phosphate Measurement for Polymerase Elongation

Han Yang  1   2 , Xiaoxin Ji  1   2 , Xiao Li  1   2 , Yunran Feng  1   2 , Ke Zhang  3   4   5 , Xuemin Guo  3   4   5 , Zhixiong Zhong  3   4   5 , Xin Mu  1   2
Affiliations

Colorimetry-Based Phosphate Measurement for Polymerase Elongation

Han Yang et al. Biomed Res Int. .

Abstract

DNA detection, which includes the measurement of variants in sequences or the presence of certain genes, is widely used in research and clinical diagnosis. Both require DNA-dependent DNA polymerase-catalyzed strand extension. Currently, these techniques rely heavily on the instruments used to visualize the results. This study introduced a simple and direct colorimetric method to measure polymerase-directed elongation. First, pyrophosphate (PPi), a by-product of strand extension, is converted into phosphate (Pi). Phosphate levels were measured using either Mo-Sb or BIOMOL Green reagent. This study showed that this colorimetry can distinguish single-base variants and detect PCR products in preset stringent conditions, implicating the potential value of this strategy to detect DNA.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
BIOMOL® Green reagent quantitatively detects single base variant. (a, b) Pi measurement using BIOMOL® Green and native PAGE analysis of samples following protocols in Figure 4(i). (c) Pi measurement using primers ended with G, C, A, or T (∗G, ∗C, ∗A, or ∗T as labeled). (d, e) Optimizing the annealing and extension conditions using primers ended with G, A, or T. (-): no Pfu in the reaction. All changed conditions are highlighted in red. (f) Pi measurement using template c.365T and its corresponding primers ended with four nucleotides, following conditions described in Figure 1(e). (g) Pi measurement using two sets of templates and primers, the revised extension condition is highlighted in red. (h) Pi measurement using 1 μM of primer and different concentration of template c.341G, following conditions described in Figure 1(g). (i) Pi measurement using 0.5 μM of template c.341G and different concentration of primers ended with G or C, following conditions described in Figure 1(g). (j) Pi measurement using 1 μM of primer and 0.5 μM of template c.341G for extension, at different cycle numbers, following conditions described in Figure 1(g). (k) Pi measurement using 1 μM of primer and changed concentration of template c.341G for extension, at different cycle numbers, following conditions described in Figure 1(g). The ratio of template to primer is shown on the right. For all c.341G samples, ∗C, ∗A, and ∗T were calculating statistic values to ∗G. For all c.365T samples, ∗G, ∗C, and A were calculating statistic values to ∗T. Data shown are mean ± S.D., n = 3 independent experiments. ns: none-statistic significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
Figure 2
Figure 2
BIOMOL® Green measurement detects strand extension in streptavidin-biotin affinity purification and PCR samples. (a) Schematic diagram of the streptavidin magnetic beads binding to biotin-labeled oligos. An adaptor oligo anneals to primers (RV-primers) through its reverse complementary sequence (RV-) and binds to beads via streptavidin. The rest sequence of RV-primers selectively binds to template for strand extension. (b) Native PAGE analysis of annealing of adaptor, RV-primer, and different templates. Red arrow indicates the template: RV-primer adaptor complex. Blue arrow indicates adaptor: RV-primer duplex. (c) Tests of influence of adaptor and the RV sequence on single base extension specificity, without the use of streptavidin beads. Assays were done using conditions described in Figure 1(g). (d) Flowchart of Pi detection coupling with streptavidin-biotin affinity purification. (e, f) Purification of streptavidin beads bound to DNAs indicated are either mock treated or boiled before Pfu-driven extension. Boiled: incubate beads at 95°C for 5 min after binding. Template c.341G (e) and c.365T (f) were tested using specific primers, respectively, ended with G, C, A, or T. (g) Detection of PCR products using plasmid as template by agarose gel analysis and BIOMOL® Green measurement following PPase treatment, respectively. Ctrl.P: control plasmid; TGT.P: target plasmid. (h, i) Detection of PCR products using HeLa genomic DNA as template. The products were analyzed by both agarose gel and BIOMOL® Green measurement following PPase treatment, respectively. For all c.341G samples, ∗C, ∗A, and ∗T were calculating statistic values to ∗G. For all c.365T samples, ∗g, ∗c, and ∗a were calculating statistic values to ∗t. For all c.542 samples, ∗C, ∗A, and ∗T were calculating statistic values to ∗G. Data shown are mean ± S.D. ns: none-statistic significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.
Figure 3
Figure 3
Principle of phosphorothioate-based strand extension detection. (a) Structure diagram of phosphorothioate. (b) Schematic diagram of phosphorothioate- (PT-modified-) based singe base extension. The high-fidelity DNA polymerase encodes 3 to 5 exonuclease activity and cleaves mismatching nucleotide during extension. PT-modification impedes such 3-5 exonuclease activity resulting in stalling of DNA polymerase. Red strand represents template. The shorter blue strand represents primer. Yellow circle with an asterisk inside represents PT-modification. (c) Schematic diagram of the measurement of Pi. PPase: pyrophosphatase. (d) Sequences of templates and matched primers. Template c.341G anneals to primer ∗G. Template c.365T anneals to primer ∗t. The asterisk represents PT-modification. Bases to be extended are highlighted in red.
Figure 4
Figure 4
Mo-Sb colorimetry detects single base variant. (a) Native-PAGE analysis of extension on template c.341G using primers ended with G or C (∗G or ∗C as labeled). (b–h) Analysis of different assay conditions for extension using the Mo-Sb colorimetric method. (b) Annealing conditions: after heating mixed DNAs at 94°C for 5 min, they were transferred into boiled water for cooling down, thermal cycler to cooled down to 4°C using default setting or to 37°C at 0.1°C per min. (c) dNTP concentrations. (d) Pfu polymerase extension temperatures. (e) PPase concentrations. (f) PPase incubation time. (g) The length of template c.341G. (h) EDTA in annealing buffer. (i) A summarized flowchart of strand extension and Pi detection by Mo-Sb colorimetry.
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