Polymerase chain reaction with nearby primers

Гарафутдинов, Р.Р.; Galimova, A. A.; Сахабутдинова, А. Р.

doi:10.1016/j.ab.2016.11.017

Cited by 10 publications

(3 citation statements)

References 29 publications

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“…Even primers designed using mathematical models sometimes form nonspecific products in negative controls and require PCR optimization. 37,38 As an initial trial, the nanopore filter experiment was performed using six different types of ssDNA, DNA #1−6, under conventional conditions for nanopore sensing. Clear blocking signals of ssDNA (160 nM) translocation events were observed for the buffer condition of 1 M KCl, 10 mM MOPS, pH 7.0.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore

et al. 2023

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This paper describes a method for the real-time counting and extraction of DNA molecules at the single-molecule level by nanopore technology. As a powerful tool for electrochemical single-molecule detection, nanopore technology eliminates the need for labeling or partitioning sample solutions at the femtoliter level. Here, we attempt to develop a DNA filtering system utilizing an α-hemolysin (αHL) nanopore. This system comprises two droplets, one filling with and one emptying DNA molecules, separated by a planar lipid bilayer containing αHL nanopores. The translocation of DNA through the nanopores is observed by measuring the channel current, and the number of translocated molecules can also be verified by quantitative polymerase chain reaction (qPCR). However, we found that the issue of contamination seems to be an almost insolvable problem in single-molecule counting. To tackle this problem, we tried to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method. Although further efforts are still needed to achieve a singlemolecule filter with electrical counting, our proposed method shows a linear relationship between the electrical counting and qPCR estimation of the number of DNA molecules.

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Section: ■ Results and Discussionmentioning

confidence: 99%

“…When using DNA #1, #2, #3, #4, #5, and #6 at low concentrations of less than 10 5 molecules, nonspecific amplification such as primer dimers was observed, and we did not obtain linear calibration curves. Even primers designed using mathematical models sometimes form nonspecific products in negative controls and require PCR optimization. , …”

Section: Resultsmentioning

confidence: 99%

Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore

et al. 2023

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“…We believe that amplification failures arise due to RNA degradation can be avoided when nearby primers are used. Recently, the higher specificity and sensitivity of PCR with nearby primers as well as high reliability of degraded DNA amplification were demonstrated [ 33 ]. In this study, to evaluate the effect of primers disposition on efficiency of RNA amplification, two primer pairs were designed as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%