Analog bases are chemically modified variants of the natural nucleotides—adenine (A), cytosine (C), guanine (G), and thymine (T)—that retain their ability to form complementary base pairs while exhibiting distinct chemical and structural properties. Within sequencing workflows, such modifications can minimize amplification bias, improve detection through enhanced fluorescent signaling, or serve as molecular markers for identifying DNA damage or base modifications.
Although the term analog bases traditionally refers to synthetically engineered nucleotides, current NGS applications also encompass naturally occurring base modifications, such as methylation and oxidation, which play pivotal roles in epigenetic gene regulation and genomic stability.
| Analog | Parent Base | Function / Use in NGS Context |
| 7-Deaza-dG/dA | Guanine / Adenine | Reduces secondary structures (e.g., G-quadruplexes), improves PCR in GC-rich regions |
| 8-Oxo-dG | Guanine | Oxidative DNA damage marker; may mispair with A → studied in variant calling, damage detection |
| 5-Methyl-dC | Cytosine | Epigenetic mark; detected in WGBS (whole-genome bisulfite sequencing) |
| 5-Hydroxymethyl-dC | Cytosine | Another epigenetic modification; differentiated via oxBS-seq, TAB-seq |
| BrdU (Bromodeoxyuridine) | Thymidine | Used in cell proliferation studies; occasionally tracked with NGS or IP-seq |
| EdU (Ethynyldeoxyuridine) | Thymidine | Clickable analog for labeling replicating DNA – useful in sequencing-based cell cycle studies |
| dU (Deoxyuridine) | Thymidine (substitute) | Found in UDG-based DNA repair and damage-seq workflows |
| dI (Deoxyinosine) | Purine analog | Can pair with multiple bases – sometimes used in degenerate primers (not common in final libraries) |
| Biotin-dUTP | Thymidine | Used for labeled DNA fragments, e.g., in ChIP-seq, pull-down assays |
| 6-MI (6-Methylisoxanthopterin) | Guanine | Fluorescent analog – sometimes used in structural or kinetic NGS assays |
Amplifying GC-rich or structurally complex DNA regions poses a significant technical challenge due to the formation of stable secondary structures, such as G-quadruplexes. Analogs like 7-deaza-dG and 7-deaza-dA suppress the formation of these structures, thereby enhancing PCR efficiency and ensuring more uniform coverage of difficult-to-amplify genomic regions.2. Detecting DNA Damage and Repair.
The oxidative analog 8-oxo-dG serves as a key biomarker of DNA damage, as it tends to mispair with adenine. Detection of this analog within sequencing experiments enables monitoring of oxidative stress, mutagenic processes, and DNA repair mechanisms—critical for understanding molecular disease mechanisms and assessing the integrity of biological samples.
Cytosine analogs such as 5-methyl-2′-deoxycytidine (5-methyl-dC) and 5-hydroxymethyl-2′-deoxycytidine (5-hydroxymethyl-dC) underpin advanced epigenomic sequencing methodologies. Techniques like whole-genome bisulfite sequencing (WGBS) and oxidative bisulfite sequencing (oxBS-seq) enable the discrimination of specific cytosine modifications and detailed mapping of methylation patterns at single-base resolution. These approaches significantly advance our understanding of epigenetic mechanisms that govern cell identity and pathophysiological states.
Thymidine analogs—most notably bromodeoxyuridine (BrdU) and ethynyldeoxyuridine (EdU)—are incorporated into newly synthesized DNA strands, marking actively replicating regions. When combined with sequencing or immunoprecipitation-based approaches, these analogs allow genome-wide tracking of replication timing and cell-cycle dynamics. Such analyses are vital for biomedical research and the development of therapeutic strategies.
Analogs such as biotin-deoxyuridine triphosphate (biotin-dUTP) and the fluorescent probe 6-methylisoxanthopterin (6-MI) enable the introduction of functional or fluorescent tags into DNA molecules. These modifications are widely employed in pull-down assays (e.g., ChIP-seq) and real-time fluorescence studies, contributing to the advancement of biochemical and structural analyses.
Analog bases represent a powerful tool for extending the analytical scope of next-generation sequencing. They not only enhance the precision and efficiency of sequencing methodologies but also provide deeper insights into the structural, epigenetic, and functional aspects of the genome. Their integration into modern sequencing protocols opens new perspectives in genomics, epigenetics, and molecular medicine.
5-Br-dU May be attached anywhere within the oligonucleotide sequence except for the 3' end. It is used for crystallographic studies of oligonucleotide structures. 5-Br-dC is photolabile, the feature which is utilized in cross-linking studies investigating protein-DNA complex structures.
5-Me-dC Increases duplex stability and is suitable for antisense applications. May be attached anywhere within the oligonucleotide sequence.
7-Deaza-8-aza-dG It is used to study oligonucleotide activity changes following alteration of the main structural component. 7-Deaza-8-aza-dG may be attached anywhere within the oligonucleotide sequence except for the 3' end.
NGS Primers Introducing our cutting-edge NGS oligonucleotide purification technology that delivers exceptional purity and minimizes cross-contamination. Our innovative methodology is designed to meet the demanding requirements of next-generation sequencing laboratories, ensuring maximum accuracy and reliability of your results.
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