Successful DNA damage repair assays depend on carefully designed oligonucleotides
that incorporate relevant lesions, matched with the correct detection method.
Bio-Synthesis provides design consultation and custom synthesis of lesion-containing oligos for
NER, BER, and UV-damage assays.
I. Nucleotide Excision Repair (NER)
NER is typically studied using synthetic oligo duplexes that carry bulky adducts
(e.g., CPDs or cisplatin-type lesions). Standard in vitro assays employ cell-free extracts
supplemented with six recombinant NER factors (RPA, XPA, XPC, TFIIH, XPG, XPF). These systems
allow monitoring of lesion recognition, dual incision, and resynthesis steps.
II. Base Excision Repair (BER)
BER assays generally use duplex oligos containing lesions such as 8-oxo-dG,
thymidine glycol, or uracil analogs. Whole-cell extracts or
purified enzymes are used to follow glycosylase activity, AP endonuclease incision, and gap filling.
BER can also be monitored in vivo using reporter constructs or damage-specific probes.
III. UV-Induced DNA Damage
UV lesions, including cyclobutane pyrimidine dimers (CPDs) and
6,4-photoProducts, are mapped using PCR-based methods such as
ligation-mediated PCR (LMPCR). For repetitive regions like telomeres,
immunoprecipitation of damaged DNA (IPoD) is preferred, enabling study of
repair kinetics in otherwise intractable sequences.
Assay considerations:
- Match lesion type to the repair pathway under investigation (BER vs NER vs UV-damage repair).
- Use appropriate flanking sequence context—GC-rich vs AT-rich regions can alter repair efficiency.
- Include controls: lesion-free duplexes and competitor substrates improve data interpretation.
- Consider in vitro vs in vivo readouts depending on your biological question.
References: Reardon & Sancar, 2003; Prakash & Prakash, 2000; Asagoshi et al., 2010;
Sundaresakumar, 2009; Tornaletti & Pfeifer, 1996; Rochette & Brash, 2010.