Bioscience Methods 2024, Vol.15, No.3, 91-101 http://bioscipublisher.com/index.php/bm 95 Figure 2 Mobile phone microscopy-based targeted DNA sequencing (Adopted from Kühnemund et al., 2017) Image caption: (a) Amplified single-molecule detection through RCA and mobile phone microscopy: selected regions within images of 10 pM to 10 fM RCPs are depicted. Scale bar, 50 mm. (b) Quantification of RCPs generated from a log10 dilution series of synthetic circular templates. Error bars: 1 s.d. from the mean, n ¼ 3; linear regression is plotted as straight line. (c) KRAS wild type and (d) KRAS mutant (codon 12 mutation) synthetic DNA fragments were circularized through selector probes, amplified through RCA, and the first base in codon 12 sequenced by unchained SBL chemistry. Sequencing reactions were imaged at both of the fluorescent channels using the mobile phone microscope and these channels were digitally superimposed. Scale bar, 20 mm. (e) Synthetic KRAS fragments at a ratio of 1:1 000 mutant/wild type were sequenced and the reaction imaged through mobile phone microscopy. Cy3 stained RCPs (wild type-base G, green bar) and Cy5 stained RCPs (mutant-base A, red bar) were quantified and plotted in the inset graph. Red arrows in the mobile phone image point to RCPs that show a Cy5 sequencing signal. Scale bar, 200 mm. (f) Mobile phone microscopybased sequence analysis: (i) fluorescence imaging of sequencing reaction, and (ii) base calling through a custom-written automated image analysis algorithm. Scale bar, 50 mm. (g) Quantification of sequencing experiments of extracted genomic DNA from cell lines and colon tumour biopsies using our mobile phone microscope. Relative frequencies of mutant (red bars) and wild type (green bars) RCPs are plotted (Adopted from Kühnemund et al., 2017) 4.4 Portable and point-of-care diagnostic tools The development of portable and point-of-care (POC) diagnostic tools has been a game-changer in the field of molecular diagnostics. Advances in miniaturization, nanotechnology, and microfluidics have led to the creation of low-cost, user-friendly, and highly sensitive POC devices. These devices often integrate various biosensing platforms with smartphone technology, enabling accurate on-site diagnostics (Zarei et al., 2017). Additionally, paper-based nucleic acid testing (NAT) platforms have emerged as robust, cost-effective, and user-friendly tools for rapid diagnostics in resource-limited settings (Choi et al., 2015). The use of CRISPR-based diagnostics, such as the SHERLOCK platform, has further enhanced the portability and accuracy of POC tools, allowing for the detection of specific genetic signatures of pathogens directly from body fluids (Stower, 2018). In summary, the technological advancements in PCR technology, sequencing platforms, AI integration, and portable
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