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  • 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
    DNA
    (plasmid or genomic)

    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