Abstract
The generation of DNA aptamer by Systematic Evolution of Ligands by Exponential Enrichment requires a good method of ssDNA generation. There are various methods developed to generate ssDNA such as streptavidin-biotin based separation techniques, asymmetric PCR and strand separation of the PCR product containing primer with a terminator and an extension of 20 nucleotides on denaturing urea-polyacrylamide gel. In the present investigation, we have shown the possible improvements for the regular lambda nuclease digestion under optimized conditions. Optimization of the PCR cycles, time course studies on lambda nuclease digestion and purification of the ssDNA from the lambda exonuclease digestion mixture was found to be able to recover ssDNA amounting up to 39.19 ± 2.48 % of the starting amount of dsDNA. These strategies can be applied to the techniques involving essential usage of ssDNA.
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Acknowledgments
We would like to thank Universiti Sains Malaysia for awarding postgraduate fellowship to Citartan for this work and the support rendered by the Ministry of Science, Technology and Innovation (MOSTI), under Brain Gain Malaysia, for the travelling and subsistence of Dr. Subash C.B. Gopinath to Malaysia.
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Supplementary Fig. 1. Schematic flow for complete optimization processes carried out. (PDF 13 kb)
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Supplementary Table 1. Statistical analysis of percentage of recovery of ssDNA after lambda exonuclease digestion (PDF 13 kb)
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Citartan, M., Tang, TH., Tan, SC. et al. Conditions optimized for the preparation of single-stranded DNA (ssDNA) employing lambda exonuclease digestion in generating DNA aptamer. World J Microbiol Biotechnol 27, 1167–1173 (2011). https://doi.org/10.1007/s11274-010-0563-8
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DOI: https://doi.org/10.1007/s11274-010-0563-8