GC-rich DNA regions are still a challenge in amplification and sequencing applications due to the high thermal and structural stability of the G-C bond promoting complex secondary structure formation.
Strand-destabilizing nucleotide analogs such as 7-Deaza-dGTP and N4-Methyl-dCTP have been widely used to overcome such amplification and sequencing challenges of GC-rich DNA regions: They are enzymatically incorporated instead of their natural counterparts (dGTP or dCTP, respectively) thereby leading to enhanced amplification, improved resolution and reduced artifacts [1-7].
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[2] Barr et al. (1986) 7-Deaza-2'-Deoxyguanosine-5'-Triphosphate: Enhanced Resolution in M13 Dideoxy Sequencing. BioTechniques 4:428.
[3] Peng et al. (2020) A novel target enrichment strategy in next-generation sequencing through 7-deaza-dGTP resistant enzymatic digestion. BMC Research Notes 13(445): https://doi.org/10.1186/s13104-020-05292-y.
[4] Motz et al. (2000) Improved Cycle Sequencing of GC-Rich Templates by a Combination of Nucleotide Analogs. BioTechniques 29: 268.
[5] Flores-Juarez et al. (2016) PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2'-deoxycytidine-5'-triphosphate. Biotechniques 61(4):175.
[6] Flores-Juarez et al. (2013) Capacity of N4-methyl-2'-deoxycytidine 5'-triphosphate to sustain the polymerase chain reaction using various thermostable DNA polymerases. Analytical Biochemistry 438(1):73.
[7] Li et al. (1993) Elimination of band compression in sequencing gels by the use of N4-methyl-2'-deoxycytidine 5'-triphosphate. Analytical Biochemistry 21(11):2709.
