Biomics

Polymerase chain reaction (PCR) is the main and widely used method for nucleic acids amplification. PCR, which developed almost 40 years ago, has undergone significant development. Different variations and significant modifications of the method were also proposed. However, despite the achievements, the improvement of PCR, due to the need to solve new complex problems, continues. One way to increase the specificity and sensitivity of PCR is to add low molecular weight compounds, so-called PCR enhancers, among which dimethyl sulfoxide (DMSO) is the most popular. In this study, we describe for the first time the possibility of using heavy water (D2O), as a PCR enhancer. It has been shown that D2O affects on PCR efficiency when the content of heavy water in the reaction mixture exceeds 50% and during amplification of relatively extended GC-rich nucleotide sequences.

polymerase chain reaction (PCR), nucleic acids, heavy water, PCR efficiency

1. Behlke M.A., Berghof-Jäger K., Brown T., et al. Polymerase Chain Reaction: Theory and Technology, Caister Academic Press, 2019.

2. Bookstein R., Lai C.C., To H., Lee W.H. PCRbased detection of a polymorphic BamHI site in intron 1 of the human retinoblastoma (RB) gene. Nucleic Acids Res. 1990. V. 18. P. 1666. doi: 10.1093/nar/18.6.1666

3. Chakrabarti R., Schutt C.E. Novel sulfoxides facilitate GC-rich template amplification. Biotechniques. 2002. V. 32. P. 866-874. doi: org/10.2144/02324rr04

4. Chakrabarti R., Schutt C.E. The enhancement of PCR amplification by low molecular-weight sulfones. Gene. 2001. V. 274. P. 293-298. doi: 10.1016/S0378-1119(01)00621-7

5. Chemeris A.V., Chemeris D.A., Magdanov E.G., Garafutdinov R.R., Nagaev N.R., Vakhitov V.A. Causes of false-negative PCR and prevention of some of them.Biomics. 2012. V. 4. No. 1. P. 31-47. (In Russian)

6. Chemeris A.V., Magdanov E.G., Garafutdinov R.R., Vakhitov V.A. How to exclude the appearance of false-positive results during the polymerase chain reaction? Vestn. biotechnol. fiz.-chem. biol. 2012. V. 8. No. 3. P. 34-45. (In Russian)

7. Chemeris D.A., Magdanov E.G., Mashkov O.I., Garafutdinov R.R., Chemeris A.V. PCR with delayed (hot or delayed) start. Biomics. 2011. V. 2. No. 1. P. 1-8. (In Russian)

8. Chevet E., Lemaître G., Katinka M.D. Low concentrations of tetramethylammonium chloride increase yield and specificity of PCR. Nucleic Acids Res. 1995. V. 23. P. 3343-3344. doi: 10.1093/nar/23.16.3343

9. Demeke T., Adams R.P. The effects of plant polysaccharides and buffer additives on PCR. Biotechniques. 1992. V. 12. P. 332-334.

10. Efimova Y.M., Haemers S., Wierczinski B., Norde W., van Well A.A. Stability of globular proteins in H2O and D2O. Biopolymers. 2007. V. 85(3). P. 264-273. doi: 10.1002/bip.20645

11. Garafutdinov R.R., Galimova A.A., Sakhabutdinova A.R., Vakhitov V.A., Chemeris A.V. PCR amplification of DNA using abutting primers. Mol. biol. 2015. V. 49. No. 4. P. 560-568. doi: 10.1134/S0026893315040056

12. Garafutdinov R.R., Galimova А.А., Sakhabutdinova А.R. Polymerase chain reaction with nearby primers. Anal. Biochem. 2017. V. 518. P. 126-133. doi: 10.1016/j.ab.2016.11.017

13. Garafutdinov R.R., Sakhabutdinova A.R., Chemeris A.V. Long-term storage of DNA molecules at room temperature. Biomics. 2020. V. 12. No. 4. P. 552-563. doi: 10.31301/2221-6197.bmcs.2020-49 (In Russian)

14. Green M.R., Sambrook J. Polymerase Chain Reaction (PCR) Amplification of GC-Rich Templates, Cold Spring Harbour Protoc. 2019. doi: 10.1101/pdb.prot095141

15. Henke W., Herdel K., Jung K., Schnorr D., Loening S.A. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 1997. V. 25. P. 3957-3958. doi: 10.1093/nar/25.19.3957

16. http://idtdna.com/

17. https://ru.wikipedia.org/wiki/%D0%A2%D1%8F%D0%B6%D1%91%D0%BB%D0%B0%D1%8F_%D0%B2%D0%BE%D0%B4%D0%B0 

18. https://www.ncbi.nlm.nih.gov/

19. Khasanova A.A., Kireeva D.R., Gibadullina N.N., Fazletdinova Z.N., Sakhabutdinova A.R., Garafutdinov R.R. Effect of hexahydropyrimidine derivatives, bis(1,2,3,4-tetrahydropyridine) and tetrahydropyrimidinium salts on the polymerase chain reaction. Biomics. 2019. V. 11. No. 1. P. 14-22. doi: 10.31301/2221-6197.bmcs.2019-03 (In Russian)

20. Korb J.P., Goddard Y., Pajski J., Diakova G., Bryant R.G. Extreme-values statistics and dynamics of water at protein interfaces. J. Phys. Chem. B. 2011. V. 115(44). P. 12845-12858. doi: 10.1021/jp2053426

21. Kreader C.A. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 1996. V. 62. P. 1102-1106.

22. Kurz M. Compatible solute influence on nucleic acids: many questions but few answers. Saline Syst. 2008. V. 4. 6. doi: 10.1186/1746-1448-4-6

23. Larsson U. Polymerization and gelation of fibrinogen in D2O. Eur. J. Biochem. 1988. V. 174(1). P. 139-144. doi: 10.1111/j.1432-1033.1988.tb14073.x

24. Makhatadze G.I., Clore G.M., Gronenborn A.M. Solvent isotope effect and protein stability. Nat. Struct. Biol. 1995. V. 2(10). P. 852-855. doi: 10.1038/nsb1095-852

25. Meyer S., Schröter M.-A., Hahn M.B., Solomun T., Sturm H., Kunte H.J. Ectoine can enhance structural changes in DNA in vitro. Sci. Rep. 2017. V. 7. 7170. doi: 10.1038/s41598-017-07441-z

26. Saiki R.K., Scharf S., Faloona F., Mullis K.B., Horn G.T., Erlich H.A., Arnheim N., Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985. V. 230. P. 1350-1354. doi: 10.1126/science.2999980

27. Sakhabutdinova A.R., Chemeris A.V., Garafutdinov R.R. Enhancement of PCR efficiency using mono- and disaccharides. Anal. Biochem. 2020. V. 606. 113858. doi: 10.1016/j.ab.2020.113858

28. Sarkar G., Kapelner S., Sommer S.S. Formamide can dramatically improve the specificity of PCR. Nucleic Acids Res. 1990. V. 18. P. 7465. doi: 10.1093/nar/18.24.7465

29. Spiess A.N., Mueller N., Ivell R. Trehalose is a potent PCR enhancer: lowering of DNA melting temperature and thermal stabilization of taq polymerase by the disaccharide trehalose. Clin. Chem. 2004. V. 50. P. 1256-1259. doi: 10.1373/clinchem.2004.031336

30. Turner S.L., Jenkins F.J. Use of deoxyinosine in PCR to improve amplification of GC-rich DNA. Biotechniques. 1995. V. 19. P. 48-52.

31. Wilson I.G. Inhibition and Facilitation of Nucleic Acid Amplification. Appl. Environ. Microbiol. 1997. V. 63. P. 3741-3751.

32. Zhang Z., Yang X., Meng L., Liu F., Shen C., Yang W. Enhanced amplification of GC-rich DNA with two organic reagents. Biotechniques. 2009. V. 47. P. 775-779. doi: 10.2144/000113203. 27