Whole genome amplification of small amounts of DNA to determine the molecular karyotype of cells
DOI:
https://doi.org/10.33910/2687-1270-2023-4-3-324-334Keywords:
degenerate primers, multiple-strand displacement amplification (MDA), polymerase chain reaction (PCR), single cell DNA sequencing, copy number variation (CNV), molecular karyotypeAbstract
Molecular genetic studies often face the need to perform uniform scaling of extremely small amounts of DNA from single cells. This, however, requires the development of specialized methods. Over the past 30 years, various approaches based on the use of degenerate primers have been proposed. However, the methods are not devoid of limitations. These limitations, among other things, are determined by the properties of polymerases. In the present study, we propose the use of two-step whole genome amplification to obtain reliable data on the molecular karyotype of the original sample based on the analysis of 20 picograms of DNA. In the protocol, at the first stage of DNA fragment flanking and amplification with the displacement of the second strand, a pair of partially degenerate primers was used: 5’-TGTGTTGGGTGTGTTTGGNNNNNNGG and 5’-TGTGTTGGGTGTGTTTGGNNNNNNTTT. At the second stage of the polymerase chain reaction, the use of a primer based on the constitutive part is sufficient: 5’-TGTGTTGGGTGTGTTTGG. The article provides reaction conditions and compares the use of various polymerases and their combinations. It discusses the possibility of using a blend of Bst and Pfu polymerases in the presence of 10 mM magnesium ions for the first stage. It also shows the potential for using Klentaq1 polymerase carrying the D732N substitution for the development of whole genome amplification methods. It has been demonstrated that the proposed method allows scaling the initial extremely small amount of DNA to obtain a sample suitable for analysis by massive parallel sequencing (next generation sequencing, NGS) using standard commercial data interpretation protocols.
References
ЛИТЕРАТУРА
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REFERENCES
Aliotta, J. M., Pelletier, J. J., Ware, J. L. et al. (1996) Thermostable Bst DNA polymerase I lacks a 3’→5’ proofreading exonuclease activity. Genetic Analysis: Biomolecular Engineering, vol. 12, no. 5–6, pp. 185–195. https://doi.org/10.1016/S1050-3862(96)80005-2 (In English)
Aviel-Ronen, S., Qi Zhu, C., Coe, B. P. et al. (2006) Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues. BMC Genomics, vol. 7, no. 1, article 312. https://doi.org/10.1186/1471-2164-7-312 (In English)
Barnes, W. M., Zhang, Z., Kermekchiev, M. B. (2021) A single amino acid change to Taq DNA polymerase enables faster PCR, reverse transcription and strand-displacement. Frontiers in Bioengineering and Biotechnology, vol. 8, article 553474. https://doi.org/10.3389/fbioe.2020.553474 (In English)
Blanco, L., Bernad, A., Lázaro, J. M. et al (1989) Highly efficient DNA synthesis by the phage φ29 DNA polymerase: Symmetrical mode of DNA replication. The Journal of Biological Chemistry, vol. 264, no. 15, pp. 8935–8940. https://doi.org/10.1016/S0021-9258(18)81883-X (In English)
Klenow, H., Henningsen, I. (1970) Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Proceedings of the National Academy of Sciences of the United States of America, vol. 65, no. 1, pp. 168–175. https://doi.org/10.1073/pnas.65.1.168 (In English)
Linck, L, Resch-Genger, U. (2010) Identification of efficient fluorophores for the direct labeling of DNA via rolling circle amplification (RCA) polymerase φ29. European Journal of Medicinal Chemistry, vol. 45, no. 12, pp. 5561– 5566. https://doi.org/10.1016/j.ejmech.2010.09.005 (In English)
Lundberg, K. S., Shoemaker, D. D., Adams, M. W. W. et al. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene, vol. 108, no. 1, pp. 1–6. https://doi.org/10.1016/0378-1119(91)90480-Y (In English)
Olszewski, M., Śpibida, M., Bilek, M., Krawczyk, B. (2017) Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans — Expression and characterization. PLoS ONE, vol. 12, no. 9, article e0184162. http://doi.org/10.1371/journal.pone.0184162 (In English)
Oscorbin, I. P., Boyarskikh, U. A., Filipenko, M. L. (2015) Large fragment of DNA polymerase I from Geobacillus sp. 777: Cloning and comparison with DNA polymerases I in practical applications. Molecular Biotechnology, vol. 57, no. 10, pp. 947–959. https://doi.org/10.1007/s12033-015-9886-x (In English)
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Saiki, R. K., Gelfand, D. H., Stoffel, S. et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, vol. 239, no. 4839, pp. 487–491. https://doi.org/10.1126/science.2448875 (In English)
Telenius, H., Carter, N. P., Bebb, C. E. et al. (1992) Degenerate oligonucleotide-primed PCR: General amplification of target DNA by a single degenerate primer. Genomics, vol. 13, no. 3, pp. 718–725. https://doi.org/10.1016/0888-7543(92)90147-K (In English)
Zong, C., Lu, S., Chapman, A. R., Xie, X. S. (2012) Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science, vol. 338, no. 6114, pp. 1622–1626. https://doi.org/10.1126/science.1229164 (In English)
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