Combing of picogram level DNA equivalent to genomic DNA present in single human cell by self propelled droplet motion over a stable gradient surface

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dc.contributor.authorYadav, Hemendra
dc.contributor.authorAlgaonkar, Prashant S.
dc.contributor.authorChakraborty, Sudip
dc.contributor.authorRamakrishna, Wusirika
dc.date.accessioned2024-01-16T14:23:31Z
dc.date.accessioned2024-08-13T10:34:23Z
dc.date.available2024-01-16T14:23:31Z
dc.date.available2024-08-13T10:34:23Z
dc.date.issued2023-11-08T00:00:00
dc.description.abstractDNA combing is a powerful technique for studying replication profile, fork-directionality and fork velocity. At present, there is requirement of a methodology to comb DNA present in a single human cell for studying replication dynamics at early embryonic stage. In our study, a surface having dual characteristics i.e., affinity towards negatively charged single DNA molecules and a hydrophobic gradient for self propelled droplet motion of combing solution was developed. The surface was made by coating of TCOS (trichloro-octylsilane) by vapor diffusion on APTES (Aminopropyl-triethoxysilane) coated glass slides. A gradient surface having high deposition efficiency (DE) was developed on which 5 picogram DNA equivalent to genomic DNA present in one single human cell can be combed. The gradient surface was thermostable in nature having the ability to sustain boiling temperature for two hours and sustain anisotropy in 70 % ethanol for 80 h. Applicability for multiple runs was enhanced such that the surface can be used for 13�14 times. Factors associated with gradient surface are unidirectional movement of combing solution droplet over the gradient surface for combing straight DNA molecules and a longer gradient surface of more than 1 cm such that long size DNA molecules can be combed. Ellipsometry and contact angle hysteresis confirmed the presence of hydrophobic gradient. XPS (X-ray photoelectron spectroscopy) and FTIR (Fourier Transform Infrared Spectroscopy) confirmed the presence of characteristic affinity towards negatively charged DNA molecules on the gradient surface. Combing solution was optimized for increasing deposition efficiency and for increasing the applicability of gradient surface for multiple runs. High temperature of combing solution was found to increase Deposition Efficiency. Combing solution was also optimized for combing single DNA molecules over the gradient surface. Single DNA molecules were combed by reducing pH and lowering concentration of triton-X in the combing solution. Dye: bp ratio was optimized for high fluorescent intensity and low surface background. � 2023 Elsevier Inc.en_US
dc.identifier.doi10.1016/j.jcis.2023.11.025
dc.identifier.issn219797
dc.identifier.urihttps://doi.org/10.1016/j.jcis.2023.11.025
dc.identifier.urihttp://10.2.3.109/handle/32116/2955
dc.language.isoen_USen_US
dc.publisherAcademic Press Inc.en_US
dc.subjectContact angleen_US
dc.subjectDepositionen_US
dc.subjectDiffusion coatingsen_US
dc.subjectDropsen_US
dc.subjectEfficiencyen_US
dc.subjectFourier transform infrared spectroscopyen_US
dc.subjectGenesen_US
dc.subjectHydrophobicityen_US
dc.subjectX ray photoelectron spectroscopyen_US
dc.subjectDeposition efficienciesen_US
dc.subjectDNA moleculesen_US
dc.subjectDroplet motionen_US
dc.subjectGenomic DNAen_US
dc.subjectGradient surfacesen_US
dc.subjectHydrophobicsen_US
dc.subjectNegatively chargeden_US
dc.subjectPicogram levelen_US
dc.subjectSingle DNA moleculesen_US
dc.subjectSingle human cellsen_US
dc.subjectDNAen_US
dc.titleCombing of picogram level DNA equivalent to genomic DNA present in single human cell by self propelled droplet motion over a stable gradient surfaceen_US
dc.title.journalJournal of Colloid and Interface Scienceen_US
dc.typeArticleen_US
dc.type.accesstypeClosed Accessen_US

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