Low Throughput Direct Cycle Sequencing of Polymerase Chain Reaction (PCR) Products

Book chapter


Nikolaos Tairis and Zouganelis, G. 2023. Low Throughput Direct Cycle Sequencing of Polymerase Chain Reaction (PCR) Products. in: Scarlett, Gary (ed.) DNA Manipulation and Analysis New York, NY Springer. pp. 195–211
AuthorsNikolaos Tairis and Zouganelis, G.
EditorsScarlett, Gary
Abstract

Polymerase Chain Reaction (PCR) products have been traditionally characterized by cloning and cycle sequencing. However, when quick sequencing data are required, the cloning step may be omitted and PCR products can be sequenced directly. We describe here a sequencing protocol that involves the gold standard Big Dye chemistry in a low throughput format using one of the latest sequencing platforms the ABI Seqstudio. Our cycle sequencing protocol follows the following steps: (1) purification of the PCR product with a spin column-based kit; (2) quality & quantity assessment of the PCR product with the use of spectrophotometry & gel electrophoresis; (3) setup and amplification of the cycle sequencing reaction; (4) Capillary Electrophoresis; (5) Sequence Data Analysis.

KeywordsSanger sequencing; PCR; cloning and cycle sequencing
Page range 195–211
Year2023
Book titleDNA Manipulation and Analysis
PublisherSpringer
Humana
Place of publicationNew York, NY
SeriesMethods in Molecular Biology
ISBN9781071630037
9781071630044
ISSN1064-3745
1940-6029
Digital Object Identifier (DOI)https://doi.org/10.1007/978-1-0716-3004-4_15
Web address (URL)http://dx.doi.org/10.1007/978-1-0716-3004-4_15
Output statusPublished
Publication datesMar 2023
Publication process dates
Deposited17 May 2023
JournalMethods in Molecular Biology
Permalink -

https://repository.derby.ac.uk/item/9yv7y/low-throughput-direct-cycle-sequencing-of-polymerase-chain-reaction-pcr-products

  • 12
    total views
  • 0
    total downloads
  • 0
    views this month
  • 0
    downloads this month

Export as

Related outputs

Compositional constraints and selection forces dictate codon usage in human bocavirus
Khandia, R.,, Singhal, S., Kumar, U., Shende, K., Pandey, M.K., Povetkin, S.N., Rzhepakovsky, I.V., Raut, A., Alexiou, A., Dhama, K., Zouganelis, G. and Khan. A. A. 2023. Compositional constraints and selection forces dictate codon usage in human bocavirus. Journal of King Saud University - Science. 35 (6), pp. 1-11. https://doi.org/10.1016/j.jksus.2023.102726
The Efficient Activity of Glabridin and its Derivatives Against EGFR-mediated Inhibition of Breast Cancer
Marios Papadakis, Arabinda Ghosh, Debanjana Ghosh, Nobendu Mukerjee, Swastika Maitra, Padmashree Das, Abhijit Dey, Souty M.Z. Sharkawi, Georgios D. Zouganelis, Athanasios Alexiou, Somdatta Yashwant Chaudhari, Ritika Sharma, Sonali Arun Waghmare, Gaber El-saber Batiha and Zouganelis, G. 2023. The Efficient Activity of Glabridin and its Derivatives Against EGFR-mediated Inhibition of Breast Cancer. Current Medicinal Chemistry. https://doi.org/10.2174/0929867330666230303120942
Nanomaterial-induced toxicity in pathophysiological models representative of individuals with pre-existing medical conditions
Sreejesh Sreedharan, Zouganelis, G., Samantha J Drake, Gyanendra Tripathi and Ali Kermanizadeh 2022. Nanomaterial-induced toxicity in pathophysiological models representative of individuals with pre-existing medical conditions. Journal of Toxicology and Environmental Health, Part B: Critical Reviews. pp. 1-27. https://doi.org/10.1080/10937404.2022.2153456
The fractal viewpoint of tumors and nanoparticles.
Alexiou, A., Tsagkaris, C., Chatzichronis, S., Koulouris, A., Haranas, I., Gkigkitzis, I., Zouganelis, G., Mukerjee, N., Maitr, S., Jha, N. K., Batiha, G. and Amjad, M. 2022. The fractal viewpoint of tumors and nanoparticles. Current Medicinal Chemistry. 30 (3), pp. 356 - 370. https://doi.org/10.2174/0929867329666220801152347
Crinum jagus (J. Thomps. Dandy): Antioxidant and protective properties as a medicinal plant on toluene-induced oxidative stress damages in liver and kidney of rats
Salihu, M., Batiha, G.E.S., Kasozi, K.I., Zouganelis, G., Sharkawi, S.M., Ahmed, E.I., Usman, I.M., Nalugo, H., Ochieng, J.J., Ssengendo, I., Okeniran, O.S., Pius, T., Kimanje, K. R., Kegoye, E.S., Kenganzi, R. and Ssempijjai, F. 2022. Crinum jagus (J. Thomps. Dandy): Antioxidant and protective properties as a medicinal plant on toluene-induced oxidative stress damages in liver and kidney of rats. Toxicology Reports. 9, pp. 699-712. https://doi.org/10.1016/j.toxrep.2022.03.026
Development and validation of anti-human Alpha synuclein DNA aptamer using computer modelling techniques—an in silico study
Rock, M., Zouganelis, G., Belchior de Andrade, A., Drake, S., Alexiou, A., Albrakati, A., Bathia, G. E. S. and Illingworth, T. 2022. Development and validation of anti-human Alpha synuclein DNA aptamer using computer modelling techniques—an in silico study. Journal of Integrative Neuroscience. 21 (1), p. 5. https://doi.org/10.31083/j.jin2101005
Characterization and phylogeny of fungi isolated from industrial wastewater using multiple genes
Ezeonuegbu, B.A., Abdullahi, M.D., Whong, C.M., Sohunago, J.W., Kassem, H.S., Yaro, C.A., Hetta, H.F., Mostafa-Hedeab, G., Zouganelis, G. and Batiha, G.E.S 2022. Characterization and phylogeny of fungi isolated from industrial wastewater using multiple genes. Scientific Reports. 12, pp. 1-11. https://doi.org/10.1038/s41598-022-05820-9
Photoperiod-Dependent Expression of MicroRNA in Drosophila.
Pegoraro, M., Fishman, B., Zonato, V., Zouganelis, G., Francis, A., Kyriacou, C. P. and Tauber, E. 2022. Photoperiod-Dependent Expression of MicroRNA in Drosophila. International Journal of Molecular Sciences. 23 (9), pp. 1-14. https://doi.org/10.3390/ijms23094935
The Inclusion of a Matrix Metalloproteinase-9 Responsive Sequence in Self-assembled Peptide-based Brain-Targeting Nanoparticles Improves the Efficiency of Nanoparticles Crossing the Blood-Brain Barrier at Elevated MMP-9 Levels
Islam, Yamir, Ehtezazi, Parinaz, Cashmore, Andrew, Marinsalda, Elena, Leach, Andrew G., Coxon, Christopher R., Fatokun, Amos A., Sexton, Darren W., Khan, Iftikhar, Downing, James, Pluchino, Stefano, Sivakumaran, Muttuswamy, Teixido, Meritxell, Ehtezazi, Touraj and Zouganelis, George 2020. The Inclusion of a Matrix Metalloproteinase-9 Responsive Sequence in Self-assembled Peptide-based Brain-Targeting Nanoparticles Improves the Efficiency of Nanoparticles Crossing the Blood-Brain Barrier at Elevated MMP-9 Levels. Journal of Pharmaceutical Science. https://doi.org/10.1016/j.xphs.2020.12.004
An old dog and new tricks: Genetic analysis of a Tudor dog recovered from the Mary Rose wreck.
Zouganelis, George D, Ogden, Rob, Nahar, Niru, Runfola, Valeria, Bonab, Maziar, Ardalan, Arman, Radford, David, Barnett, Ross, Larson, Greger, Hildred, Alex, Jones, Mark and Scarlett, Garry 2014. An old dog and new tricks: Genetic analysis of a Tudor dog recovered from the Mary Rose wreck. Forensic Science International. 245, pp. 51-7. https://doi.org/10.1016/j.forsciint.2014.10.001
In Vivo and In Vitro Evaluation of Preventive Activity of Inflammation and Free Radical Scavenging Potential of Plant Extracts from Oldenlandia corymbosa L.
Al Bashera, M., Mosaddik, A., El-Saber Batiha, G., Alqarni, M., Islam, M.A., Zouganelis, G., Alexiou, A. and Zahan, R. 2021. In Vivo and In Vitro Evaluation of Preventive Activity of Inflammation and Free Radical Scavenging Potential of Plant Extracts from Oldenlandia corymbosa L. Applied Sciences. 11 (19), p. 9073. https://doi.org/10.3390/app11199073