Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope

Gali Adamu Ishaku

doi:doi:10.11648/j.ijbse.20190703.11

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Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope

Gali Adamu Ishaku

Published in International Journal of Biomedical Science and Engineering (Volume 7, Issue 3)

Received: 14 August 2019 Accepted: 9 September 2019 Published: 24 September 2019

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Abstract

λDNA tightrope can be referred to as λDNA is suspended between two beads above the surface of the flow cell which allows the λDNA to be viewed extended rather than in collapse form. It is use for DNA studies such DNA repairs, but when using the normal protocol (at a velocity of 300 µl/minutes, beads size of 5 µm and concentration of 500.0 µg/ml of 1.0 λDNA) it results to multiple tightropes forming on the beads which interference with a many studies. In this study, different conditions were optimized to develop a single stranded λDNA tight rope by taking into consideration the following conditions, velocity (100 to 600), beads size (3 µm and 5µm), concentration (500.0 µg/ml, 375.0 µg/ml and 250.0 µg/ml) and time. To form the tightrope, beads were added into water in an eppendorf tube, centrifuged and sonicated before putting into the flow cell. The flow cell, syringe pump and perfusion tube were all fixed together, where ABC buffer and λDNA were put into the perfusion tube and set according to protocol to make the tightropes. YOYO-1 dye was used to stain the λDNA before using atomic force microscopy to image the tightropes, the same procedure was repeated for the different conditions and all dispense readings of the flow rate were taken at not less than 3.0 µl/minutes. λDNA on the surface of the flow cell was observed at the velocity of 100 and 200 µl/minutes for all the conditions. The optimized result was at velocity of 300µl/ml, concentration 375.0 µg/ml of 1.0 μλDNA and beads size of 3 µl which lasted for 15 minutes which gave the best result of single stranded λDNA tightrope compared to all the conditions and the normal protocol.

Published in	International Journal of Biomedical Science and Engineering (Volume 7, Issue 3)
DOI	10.11648/j.ijbse.20190703.11
Page(s)	61-67
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2019. Published by Science Publishing Group

Keywords

Beads, DNA Tightrope, Flow Cell, Phage-Lambda (λDNA) and Yoyo-1 Dye

References

[1]	Kad N. M., Wang H., Kennedy G. G., Warshaw D. M., and Van Houten B., (2010). Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single-molecule imaging of quantum-dot-labeled proteins. Molecular Cell, 37: 702–713.
[2]	Gorman J., and Greene E. C. (2008). Vsisualizing one-dimensional diffusion of proteins along DNA. Nat. Struct. Mol. Biol. 15, 768–774.
[3]	Cornish P. V., and Ha T., (2007). A survey of single-molecule techniques in chemical biology. ACS Chemical Biology, 2: 53–61.
[4]	Wang J., Barnett J. T., Pollard M. R., Kad N. M. (2016). Integrating Optical Tweezers, DNA Tightropes, and Single-Molecule Fluorescence Imaging: Pitfalls and Traps. Methods in Enzymology. 1-22.
[5]	Pan H., Bilinovich M. S., Kaur P., Riehn R., Wang H. and Williams C. D., (2017). CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level. Nucleic Acids Research. 1-14.
[6]	Wu D., Kaur P., Li M. Z., Bradford C. K., Wang H., and Erie A. D. (2016) Visualizing the Path of DNA through Proteins Using DREEM Imaging. Molecular Cell 61, 315–323.
[7]	Hughes D. C., Wang H, Ghodke H., Simons M., Towheed A., Peng Y., Houten V. and Kad M. N. (2013) Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes. Nucleic Acids Research; 1–12.
[8]	Bustamante C., Bryant Z., and Smith S. B., (2003). Ten years of tension: Single-molecule DNA mechanics. Nature, 421: 423-427.
[9]	Ngwu NW, Effa EB, Ftepti BJ, Gali AI, Useh MU, Samuel CJ. (2016). Biochemical Studies of Ocimum sanctum and Olax subscorpioidea leaf extracts. British Journal of Pharmaceutical Research. 12 (4): 1-9.
[10]	Walter N. G., Huang C. Y., Manzo A. J., and Sobhy M. A., (2008). Do-it-yourself guide: How to use the modern single-molecule toolkit. Nature Methods, 5: 475-489.
[11]	Joo C., Balci H., Ishitsuka Y., Buranachai C., and Ha T., (2008). Advances in single-molecule fluorescence methods for molecular biology. Annual Review of Biochemistry, 77: 51-76.
[12]	Simons M., Pollard R. M., Hughes D. C., Ward D. A., Houten V. B., Towrie M., Botchway W. S., Parker W. A. and Kad M. N. (2015) Directly interrogating single quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Scientific Reports. 5: 18486.
[13]	Ashkin A., Dziedzic J. M., and Yamane T., (1987). Optical trapping and manipulation of single cells using infrared-laser beams. Nature, 330: 769-771.
[14]	Springall L., Hughes D. C., Simons M., Azinas S., Houten V. B. and Kad M. N. (2017). Recruitment of UvrBC complexes to UV-induced damage in the absence of UvrA increases cell survival. Nucleic Acids Research: 1-10.
[15]	Von Hippel P. H., and Berg O. G., (1989) Facilitated target location in biological systems. Journal of Biological Chemistry, 264: 675–678.
[16]	Shimamoto N., (1999). One-dimensional diffusion of proteins along DNA. Its biological and chemical significance revealed by single molecule measurements. Journal of Biological Chemistry, 274: 15293–15296.
[17]	Halford S. E., and Marko J. F., (2004). How do site-specific binding proteins find their targets? Nucleic Acids Research, 32: 3040–3052.
[18]	Spies M., Bianco P. R., Dillingham M. S., Handa N., Baskin R. J., and Kowalczykowski S. C., (2003). A molecular throttle: The recombination hotspot, χ, controls DNA translocation by the RecBCD helicase. Cell, 114: 647-654.
[19]	Kim J. H., and Larson R. G., (2007). Single-molecule analysis of 1D diffusion and transcription elongation of T7 RNA polymerase along individual stretched DNA molecules. Nucleic Acids Research, 11: 3848-3858.
[20]	Kabata H., Kurosawa O., Arai I., Washizu M., Margarson S. A., Glass R. E. and Shimamoto, N. (1993). Visualization of single molecules of RNA polymerase sliding along DNA. Science, 262: 1561-1563.
[21]	Greene E. C., and Mizuuchi K., (2002). Direct observation of single MuB polymers: evidence for a DNA-dependent conformational change for generating an active target complex. Molecular Cell, 9: 1079-1089.
[22]	Van Oijen A. M., Blainey P. C., Crampton D. J., Richardson C. C., Ellenberger T., and Xie X. S., (2003). Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder. Science, 301: 1235-1238.
[23]	Graneli A., Yeykal C. C., Prasad T. K., and Greene E. C., (2006). Organized arrays of individual DNA molecules tethered to supported lipid bilayers. Langmuir, 22: 292-299.

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APA Style

Gali Adamu Ishaku. (2019). Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope. International Journal of Biomedical Science and Engineering, 7(3), 61-67. https://doi.org/10.11648/j.ijbse.20190703.11

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ACS Style

Gali Adamu Ishaku. Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope. Int. J. Biomed. Sci. Eng. 2019, 7(3), 61-67. doi: 10.11648/j.ijbse.20190703.11

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AMA Style

Gali Adamu Ishaku. Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope. Int J Biomed Sci Eng. 2019;7(3):61-67. doi: 10.11648/j.ijbse.20190703.11

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@article{10.11648/j.ijbse.20190703.11,
  author = {Gali Adamu Ishaku},
  title = {Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope},
  journal = {International Journal of Biomedical Science and Engineering},
  volume = {7},
  number = {3},
  pages = {61-67},
  doi = {10.11648/j.ijbse.20190703.11},
  url = {https://doi.org/10.11648/j.ijbse.20190703.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijbse.20190703.11},
  abstract = {λDNA tightrope can be referred to as λDNA is suspended between two beads above the surface of the flow cell which allows the λDNA to be viewed extended rather than in collapse form. It is use for DNA studies such DNA repairs, but when using the normal protocol (at a velocity of 300 µl/minutes, beads size of 5 µm and concentration of 500.0 µg/ml of 1.0 λDNA) it results to multiple tightropes forming on the beads which interference with a many studies. In this study, different conditions were optimized to develop a single stranded λDNA tight rope by taking into consideration the following conditions, velocity (100 to 600), beads size (3 µm and 5µm), concentration (500.0 µg/ml, 375.0 µg/ml and 250.0 µg/ml) and time. To form the tightrope, beads were added into water in an eppendorf tube, centrifuged and sonicated before putting into the flow cell. The flow cell, syringe pump and perfusion tube were all fixed together, where ABC buffer and λDNA were put into the perfusion tube and set according to protocol to make the tightropes. YOYO-1 dye was used to stain the λDNA before using atomic force microscopy to image the tightropes, the same procedure was repeated for the different conditions and all dispense readings of the flow rate were taken at not less than 3.0 µl/minutes. λDNA on the surface of the flow cell was observed at the velocity of 100 and 200 µl/minutes for all the conditions. The optimized result was at velocity of 300µl/ml, concentration 375.0 µg/ml of 1.0 μλDNA and beads size of 3 µl which lasted for 15 minutes which gave the best result of single stranded λDNA tightrope compared to all the conditions and the normal protocol.},
 year = {2019}
}

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TY  - JOUR
T1  - Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope
AU  - Gali Adamu Ishaku
Y1  - 2019/09/24
PY  - 2019
N1  - https://doi.org/10.11648/j.ijbse.20190703.11
DO  - 10.11648/j.ijbse.20190703.11
T2  - International Journal of Biomedical Science and Engineering
JF  - International Journal of Biomedical Science and Engineering
JO  - International Journal of Biomedical Science and Engineering
SP  - 61
EP  - 67
PB  - Science Publishing Group
SN  - 2376-7235
UR  - https://doi.org/10.11648/j.ijbse.20190703.11
AB  - λDNA tightrope can be referred to as λDNA is suspended between two beads above the surface of the flow cell which allows the λDNA to be viewed extended rather than in collapse form. It is use for DNA studies such DNA repairs, but when using the normal protocol (at a velocity of 300 µl/minutes, beads size of 5 µm and concentration of 500.0 µg/ml of 1.0 λDNA) it results to multiple tightropes forming on the beads which interference with a many studies. In this study, different conditions were optimized to develop a single stranded λDNA tight rope by taking into consideration the following conditions, velocity (100 to 600), beads size (3 µm and 5µm), concentration (500.0 µg/ml, 375.0 µg/ml and 250.0 µg/ml) and time. To form the tightrope, beads were added into water in an eppendorf tube, centrifuged and sonicated before putting into the flow cell. The flow cell, syringe pump and perfusion tube were all fixed together, where ABC buffer and λDNA were put into the perfusion tube and set according to protocol to make the tightropes. YOYO-1 dye was used to stain the λDNA before using atomic force microscopy to image the tightropes, the same procedure was repeated for the different conditions and all dispense readings of the flow rate were taken at not less than 3.0 µl/minutes. λDNA on the surface of the flow cell was observed at the velocity of 100 and 200 µl/minutes for all the conditions. The optimized result was at velocity of 300µl/ml, concentration 375.0 µg/ml of 1.0 μλDNA and beads size of 3 µl which lasted for 15 minutes which gave the best result of single stranded λDNA tightrope compared to all the conditions and the normal protocol.
VL  - 7
IS  - 3
ER  -

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Author Information

Gali Adamu Ishaku

School of Biological Sciences, University of Essex, Essex, United Kingdom

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Plain Text BibTeX RIS

APA Style

Gali Adamu Ishaku. (2019). Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope. International Journal of Biomedical Science and Engineering, 7(3), 61-67. https://doi.org/10.11648/j.ijbse.20190703.11

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ACS Style

Gali Adamu Ishaku. Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope. Int. J. Biomed. Sci. Eng. 2019, 7(3), 61-67. doi: 10.11648/j.ijbse.20190703.11

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AMA Style

Gali Adamu Ishaku. Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope. Int J Biomed Sci Eng. 2019;7(3):61-67. doi: 10.11648/j.ijbse.20190703.11

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@article{10.11648/j.ijbse.20190703.11,
  author = {Gali Adamu Ishaku},
  title = {Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope},
  journal = {International Journal of Biomedical Science and Engineering},
  volume = {7},
  number = {3},
  pages = {61-67},
  doi = {10.11648/j.ijbse.20190703.11},
  url = {https://doi.org/10.11648/j.ijbse.20190703.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijbse.20190703.11},
  abstract = {λDNA tightrope can be referred to as λDNA is suspended between two beads above the surface of the flow cell which allows the λDNA to be viewed extended rather than in collapse form. It is use for DNA studies such DNA repairs, but when using the normal protocol (at a velocity of 300 µl/minutes, beads size of 5 µm and concentration of 500.0 µg/ml of 1.0 λDNA) it results to multiple tightropes forming on the beads which interference with a many studies. In this study, different conditions were optimized to develop a single stranded λDNA tight rope by taking into consideration the following conditions, velocity (100 to 600), beads size (3 µm and 5µm), concentration (500.0 µg/ml, 375.0 µg/ml and 250.0 µg/ml) and time. To form the tightrope, beads were added into water in an eppendorf tube, centrifuged and sonicated before putting into the flow cell. The flow cell, syringe pump and perfusion tube were all fixed together, where ABC buffer and λDNA were put into the perfusion tube and set according to protocol to make the tightropes. YOYO-1 dye was used to stain the λDNA before using atomic force microscopy to image the tightropes, the same procedure was repeated for the different conditions and all dispense readings of the flow rate were taken at not less than 3.0 µl/minutes. λDNA on the surface of the flow cell was observed at the velocity of 100 and 200 µl/minutes for all the conditions. The optimized result was at velocity of 300µl/ml, concentration 375.0 µg/ml of 1.0 μλDNA and beads size of 3 µl which lasted for 15 minutes which gave the best result of single stranded λDNA tightrope compared to all the conditions and the normal protocol.},
 year = {2019}
}

Copy | Download

TY  - JOUR
T1  - Optimizing Different Conditions to Develop a Single Stranded Phage-Lambda DNA (λDNA) Tightrope
AU  - Gali Adamu Ishaku
Y1  - 2019/09/24
PY  - 2019
N1  - https://doi.org/10.11648/j.ijbse.20190703.11
DO  - 10.11648/j.ijbse.20190703.11
T2  - International Journal of Biomedical Science and Engineering
JF  - International Journal of Biomedical Science and Engineering
JO  - International Journal of Biomedical Science and Engineering
SP  - 61
EP  - 67
PB  - Science Publishing Group
SN  - 2376-7235
UR  - https://doi.org/10.11648/j.ijbse.20190703.11
AB  - λDNA tightrope can be referred to as λDNA is suspended between two beads above the surface of the flow cell which allows the λDNA to be viewed extended rather than in collapse form. It is use for DNA studies such DNA repairs, but when using the normal protocol (at a velocity of 300 µl/minutes, beads size of 5 µm and concentration of 500.0 µg/ml of 1.0 λDNA) it results to multiple tightropes forming on the beads which interference with a many studies. In this study, different conditions were optimized to develop a single stranded λDNA tight rope by taking into consideration the following conditions, velocity (100 to 600), beads size (3 µm and 5µm), concentration (500.0 µg/ml, 375.0 µg/ml and 250.0 µg/ml) and time. To form the tightrope, beads were added into water in an eppendorf tube, centrifuged and sonicated before putting into the flow cell. The flow cell, syringe pump and perfusion tube were all fixed together, where ABC buffer and λDNA were put into the perfusion tube and set according to protocol to make the tightropes. YOYO-1 dye was used to stain the λDNA before using atomic force microscopy to image the tightropes, the same procedure was repeated for the different conditions and all dispense readings of the flow rate were taken at not less than 3.0 µl/minutes. λDNA on the surface of the flow cell was observed at the velocity of 100 and 200 µl/minutes for all the conditions. The optimized result was at velocity of 300µl/ml, concentration 375.0 µg/ml of 1.0 μλDNA and beads size of 3 µl which lasted for 15 minutes which gave the best result of single stranded λDNA tightrope compared to all the conditions and the normal protocol.
VL  - 7
IS  - 3
ER  -

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