DNA Damage and PreCR
DNA is susceptible to many types of damage resulting from exposure to a variety of chemical or environmental reagents, manipulation or simply aging. The table below lists some possible DNA samples and the impact of the damage they may undergo.
Note: The extent of damage caused by exposure to different reagents can vary and its importance will depend on how the DNA is being used.
| Source of DNA | Potential Damage | Comments | References |
|---|---|---|---|
| Ancient DNA | abasic sites, deaminated cytosine, oxidized bases, fragmentation, nicks | Cytosine deamination has been reported to be the most prevalent cause of sequencing artifacts in ancient DNA. | Gilbert, M.T. et al. (2007) Nuc. Acid Res., 35, 1-10. PMID: 16920744 Hofreiter, M. et al. (2001) Nuc. Acid Res., 29, 4793. PMID: 11726688 |
|
Environmental |
fragmentation, nicks | Nicks and fragmentation can increase the formation of artifactual chimeric genes during amplification. | Qiu, X. et al. (2001) Appl. Envir. Microbiol., 67, 880. PMID: 11157258 |
| Source of Damage | |||
| Exposure to Ionizing Radiation |
abasic sites, oxidized bases, fragmentation, nicks | Ionizing radiation is used to sterilize samples. | Sutherland, B.M. et al. (2000) Biochemistry, 39, 8026. PMID: 10891084 |
| Exposure to Heat | fragmentation, nicks, abasic sites, oxidized bases, deaminated cytosine, cyclopurine lesions | Heating DNA accelerates the hydrolytic and oxidative reactions in aqueous solutions. | Bruskov, V.I. (2002) Nuc. Acids Res., 30, 1354. PMID: 11884633 |
| Phenol/ Chloroform Extraction |
oxidized bases | Guanine is more sensitive to oxidation than the other bases and forms 8-oxo-G. 8-oxo-G can base pair with A making this damage potentially mutagenic. | Finnegan, M.T. (1995) Biochem. Soc. Trans., 23, 403S. PMID: 8566318 |
| Exposure to Light (UV) |
thymine dimers, (cyclobutane pyrimidine dimers) pyrimidine (6–4) pyrimidine photo products | UV trans-illumination to visualize DNA causes thymine dimer formation. | Cadet, J. et al. (2005) Mutat. Res., 571, 3-17. PMID: 15748634 Pfeifer, G.P. et al. (2005) Mutat. Res., 571, 19-31. PMID: 15748635 |
| Mechanical Shearing |
fragmentation, nicks | Normal DNA manipulations such as pipetting or mixing can shear or nick DNA. | |
| Dessication | fragmentation, nicks, oxidized bases | Mandrioli, M. et al. (2006) Entomol. Exp. App., 120, 239. | |
| Storage in Aqueous Solution |
abasic sites, oxidized bases, deaminated cytosine, nicks, fragmentation | Long term storage in aqueous solution causes the accumulation of DNA damage. | Lindahl, T. et al. (1972) Biochemistry, 11, 3610 and 3618. PMID: 4626532 |
| Exposure to Formalin |
DNA-DNA crosslinks, DNA-protein crosslinks | Formaldehyde solution that has not been properly buffered becomes acidic, increasing abasic site formation. | Workshop on recovering DNA from formalin preserved biological samples. (2006) The National Academies Press. |
NEB now offers the PreCR Repair Mix (NEB #M0309), a blend of recombinant proteins designed to repair multiple types of damaged template DNA. It can be used in conjunction with any thermophilic polymerase.
The feature article, DNA Damage - the major cause of missing pieces from the DNA puzzle, provides a more in-depth look at the effects of different types of DNA damage.
| DNA Damage | Cause | Repaired by PreCR Repair Mix? |
|---|---|---|
| abasic sites | hydrolysis | yes |
| nicks | hydrolysis nucleases shearing |
yes |
| thymidine dimers | UV radiation | yes |
| blocked 3´-ends | multiple | yes |
| oxidized guanine | oxidation | yes |
| oxidized pyrimidines |
oxidation | yes |
| deaminated cytosine |
hydrolysis | yes |
| fragmentation | hydrolysis nucleases shearing |
no |
| Protein- DNA crosslinks | formaldehyde | no |
