Biomics

PCR of so-called "difficult" templates, including DNA regions with a high content of GC pairs, poses a certain problem. However, there is no clear boundary of the percentage of GC pairs for assigning DNA fragments to difficult targets. Nevertheless, those with a GC composition exceeding 60% can be considered as such. Moreover, the total content of GC pairs in a certain sequence may be relatively low, but there may be a regions with an extremely high GC composition locally in it. In this regard, the simple mention of the percentage of GC pairs provides only a very rough description of the templates, since the entire nucleotide sequence of the selected target makes a significant contribution to the efficiency of the amplification process, and it is different for various targets each time. Due to the fact that GC pairs form three hydrogen bonds between complementary nitrogen bases, unlike AT pairs, which have only two such bonds, GC-rich DNA fragments after the denaturation stage are much more prone to forming strong secondary structures, which DNA polymerase cannot overcome when synthesizing a new complementary chain under normal conditions. To amplify GC-rich targets, it is necessary to use various PCR enhancers that have different effects on the polymerization process of the DNA chain. A number of enhancers in the form of organic solvents reduce the melting temperature of DNA and thereby prevent the formation of secondary structures, while others reduce the effect of these solvents on the enzymatic activity of DNA polymerase, providing a synergistic effect. The use of modified analogues of dGTP or dCTP destabilizes GC pairs, depriving them of the opportunity to form secondary structures. The features of thermal cycling during PCR, in the form of subcycling at the annealing stage, as well as short-term high-temperature pulses and other temperature variations, are important. However, there is no single recipe for PCR with any GC-rich templates, and in order to achieve the desired result, it is necessary to try various enhancers and/or combinations of them, different DNA polymerases, as well as thermal cycling modes.

PCR, GC-rich target, enhancer, DMSO, betaine, glycerol, formamide, 7-deaza-dGTP, DNA polymerase

1. Agarwal RK, Perl A. PCR amplification of highly GC-rich DNA template after denaturation by NaOH. Nucleic Acids Res. 21(22). 5283-5284. doi: 10.1093/nar/21.22.5283
2. Bachmann HS, Siffert W, Frey UH. Successful amplification of extremely GC-rich promoter regions using a novel 'slowdown PCR' technique. Pharmacogenetics. 2003. 13(12). 759-766. doi: 10.1097/00008571-200312000-00006
3. Chakrabarti R, Schutt CE. Novel sulfoxides facilitate GC-rich template amplification. Biotechniques. 2002. 32(4). 866, 868, 870-872, 874. doi: 10.2144/02324rr04
4. Chakrabarti R, Schutt CE. The enhancement of PCR amplification by low molecular-weight sulfones. Gene. 2001. 274(1-2). 293-298. doi: 10.1016/s0378-1119(01)00621-7
5. Chemeris AV, Chemeris DA, Magdanov EG et al. Causes of false-negative PCR and how to avoid some of them. Biomics. 2012. 4(1). 31-47. (In Russian)
6. Chemeris DA, Magdanov EG, Mashkov OI et al. Hot start or time-release PCR. Biomics. 2011. 2(1). 1-8. (In Russian)
7. Dutton CM, Paynton C, Sommer SS. General method for amplifying regions of very high G+C content. Nucleic Acids Res. 21(12). 2953-2954. doi: 10.1093/nar/21.12.2953
8. Farell EM, Alexandre G. Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates. BMC Res Notes. 2012. 5. 257. doi: 10.1186/1756-0500-5-257
9. Flores-Juárez CR, González-Jasso E, Antaramian A et al. Capacity of N4-methyl-2'-deoxycytidine 5'-triphosphate to sustain the polymerase chain reaction using various thermostable DNA polymerases. Anal Biochem. 2013. 438(1). 73-81. doi: 10.1016/j.ab.2013.03.025
10. Flores-Juárez CR, González-Jasso E, Antaramian A et al. PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2'-deoxycytidine 5'-triphosphate. Biotechniques. 2016. 61(4). 175-182. doi: 10.2144/000114457
11. Frey UH, Bachmann HS, Peters J et al. PCR-amplification of GC-rich regions: 'slowdown PCR'. Nat Protoc. 2008. 3(8). 1312-1317. doi: 10.1038/nprot.2008.112
12. Garafutdinov RR, Chemeris DA, Sakhabutdinova AR et al. PCR enhancers. Chemical and biological substances. Biomics. 2025. 17(2). 157-169. doi: 10.31301/2221-6197.bmcs.2025-12 (In Russian)
13. Garafutdinov RR, Galimova AA, Sakhabutdinova AR. Polymerase chain reaction with nearby primers. Anal Biochem. 518. 126-133. doi: 10.1016/j.ab.2016.11.017
14. Henke W, Herdel K, Jung K et al. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 25(19). 3957-3958. doi: 10.1093/nar/25.19.3957
15. Horáková H, Polakovičová I, Shaik GM et al. 1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer. BMC Biotechnol. 2011. 11. 41. doi: 10.1186/1472-6750-11-41
16. Hubé F, Reverdiau P, Iochmann S et al. Improved PCR method for amplification of GC-rich DNA sequences. Mol Biotechnol. 2005. 31(1). 81-84. doi: 10.1385/MB:31:1:081
17. Kang J, Lee MS, Gorenstein DG. The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers. J Biochem Biophys Methods. 2005. 64(2). 147-151. doi: 10.1016/j.jbbm.2005.06.003
18. Karunanathie H, Kee PS, Ng SF et al. PCR enhancers: Types, mechanisms, and applications in long-range PCR. Biochimie. 2022. 197. 130-143. doi: 10.1016/j.biochi.2022.02.009
19. Liu Q, Sommer SS. Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: application to the inversion hotspot in the factor VIII gene. Biotechniques. 1998. 25(6). 1022-1028. doi: 10.2144/98256rr01
20. Lotte Hansen L, Justesen J. PCR Amplification of Highly GC-Rich Regions. CSH Protoc. 2006. 2006(1). pdb.prot4093. doi: 10.1101/pdb.prot4093
21. Mamedov TG, Pienaar E, Whitney SE et al. A fundamental study of the PCR amplification of GC-rich DNA templates. Comput Biol Chem. 2008. 32(6). 452-457. doi: 10.1016/j.compbiolchem.2008.07.021
22. McConlogue L, Brow MA, Innis MA. Structure-independent DNA amplification by PCR using 7-deaza-2'-deoxyguanosine. Nucleic Acids Res. 1988. 16(20). 9869. doi: 10.1093/nar/16.20.9869
23. Mousavian Z, Sadeghi HM, Sabzghabaee AM et al. Polymerase chain reaction amplification of a GC rich region by adding 1,2 propanediol. Adv Biomed Res. 2014. 3. 65. doi: 10.4103/2277-9175.125846
24. Musso M, Bocciardi R, Parodi S et al. Betaine, dimethyl sulfoxide, and 7-deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences. J Mol Diagn. 2006. 8(5). 544-550. doi: 10.2353/jmoldx.2006.060058
25. Orpana AK, Ho TH, Stenman J. Multiple heat pulses during PCR extension enabling amplification of GC-rich sequences and reducing amplification bias. Anal Chem. 84(4). 2081-2087. doi: 10.1021/ac300040j
26. Ralser M, Querfurth R, Warnatz HJ, Lehrach H, Yaspo ML, Krobitsch S. An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun. 347(3). 747-751. doi: 10.1016/j.bbrc.2006.06.151
27. Sahdev S, Saini S, Tiwari P et al. Amplification of GC-rich genes by following a combination strategy of primer design, enhancers and modified PCR cycle conditions. Mol Cell Probes. 2007. 21(4). 303-307. doi: 10.1016/j.mcp.2007.03.004
28. Sakhabutdinova AR, Chemeris AV, Garafutdinov RR. Enhancement of PCR efficiency using mono- and disaccharides. Anal Biochem. 2020. 606. 113858. doi: 10.1016/j.ab.2020.113858
29. Sakhabutdinova AR, Chemeris DA, Chemeris AV et al. PCR enhancers. I. General information. Biomics. 2023. 15(3). 218-223. doi: 10.31301/2221-6197.bmcs.2023-20 (In Russian)
30. Sakhabutdinova AR, Chemeris DA, Garafutdinov RR. Enhancers of PCR. V. Nanomaterials or nanoPCR. Biomics. 2025. 17(2). 193-205. doi: 10.31301/2221-6197.bmcs.2025-15 (In Russian)
31. Sarkar G, Kapelner S, Sommer SS. Formamide can dramatically improve the specificity of PCR. Nucleic Acids Res. 1990. 18(24). 7465. doi: 10.1093/nar/18.24.7465
32. Schnoor M, Voss P, Cullen P et al. Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer. Biochem Biophys Res Commun. 2004. 322(3). 867-672. doi: 10.1016/j.bbrc.2004.07.200
33. Schuchard M, Sarkar G, Ruesink T et al. Two-step "hot" PCR amplification of GC-rich avian c-myc sequences. Biotechniques. 1993. 14(3). 390-394.
34. Seifi T, Ghaedi K, Salamian A et al. Amplification of GC-rich Putative Mouse PeP Promoter using Betaine and DMSO in Ammonium Sulfate Polymerase Chain Reaction Buffer. Avicenna J Med Biotechnol. 2012. 4(4). 206-209.
35. Shi X, Jarvis DL. A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes. Anal Biochem. 2006. 356(2). 222-228. doi: 10.1016/j.ab.2006.06.028
36. Shi Y, Liu YL, Lai PY et al. Ionic liquids promote PCR amplification of DNA. Chem Commun (Camb). 48(43). 5325-5327. doi: 10.1039/c2cc31740k
37. Shore S, Paul N. Robust PCR Amplification of GC-Rich Targets with Hot Start 7-deaza-dGTP. BioTechniques. 2010. 49(5). 841-843. doi: 10.2144/000113552
38. Strien J, Sanft J, Mall G. Enhancement of PCR amplification of moderate GC-containing and highly GC-rich DNA sequences. Mol Biotechnol. 2013. 54(3). 1048-1054. doi: 10.1007/s12033-013-9660-x
39. Turner SL, Jenkins FJ. Use of deoxyinosine in PCR to improve amplification of GC-rich DNA. Biotechniques. 1995. 19(1). 48-52.
40. Varadaraj K, Skinner DM. Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases. Gene. 1994. 140(1). 1-5. doi: 10.1016/0378-1119(94)90723-4
41. Wei M, Deng J, Feng K et al. Universal method facilitating the amplification of extremely GC-rich DNA fragments from genomic DNA. Anal Chem. 2010. 82(14). 6303-6307. doi: 10.1021/ac100797t
42. Xie B, Chen J, Wang Z et al. Sweet enhancers of polymerase chain reaction. PLoS One. 2024. 19(10). e0311939. doi: 10.1371/journal.pone.0311939
43. Yang Z, Yang J, Yue L et al. Enhancement Effects and Mechanism Studies of Two Bismuth-Based Materials Assisted by DMSO and Glycerol in GC-Rich PCR. Molecules. 2023. 28(11). 4515. doi: 10.3390/molecules28114515
44. Zhang Z, Kermekchiev MB, Barnes WM. Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq. J Mol Diagn. 12(2). 152-161. doi: 10.2353/jmoldx.2010.090070
45. Zhang Z, Yang X, Meng L et al. Enhanced amplification of GC-rich DNA with two organic reagents. Biotechniques. 2009. 47(3). 775-779. doi: 10.2144/000113203
46. Zubov VV, Chemeris DA, Sakhabutdinova AR et al. Enhancers of PCR. III. Amplification of long templates. Biomics. 2025. 17(2). 170-181. doi: 10.31301/2221-6197.bmcs.2025-13 (In Russian)