What influences the rate of DNA degradation?
DNA degradation and DNA damage occurs through enzymatic processes,
oxidative damage, UV radiation, and hydrolysis (Schroeder et al., 2006).
DNA degradation starts within minutes or hours after sampling from a
live specimen (Graham et al., 2015), and will continue to degrade
regardless of how the DNA has been preserved (Guo et al., 2018).
Endonucleases and exonucleases can lead to the rapid break down of DNA
inside the cells. As any enzyme activity is sensitive to temperature,
the degradation process is reduced at lower temperatures. Thus keeping
samples cold will slow down the enzymatic degradation of DNA. In
addition, oxidative damage by free radicals and hydrolysis through
interaction with water (especially acidic water) compromises DNA
integrity. Tissue samples will always be, to some extent, subject to all
the processes presented above during transport. Finally, UV radiation
from direct sunlight can induce double-stranded DNA damage and form T-T
dimers.
Once extracted, DNA continues to degrade even while being stored under
optimal conditions (i.e. low temperature, buffered media, sterile
environment, and/or minimal manipulations) (Guo et al., 2018). Storing
extracted DNA in a solution that buffers the pH (e.g. Tris-HCl pH 8)
protects samples from oxidative damage and hydrolysis of phosphate
bonds, increasing the chance of retaining good DNA quality. Tris-HCl
buffering is often combined with ethylenediaminetetraacetic acid (EDTA),
commonly known as TE buffer. EDTA binds to metal ions, deactivating
metal-dependent enzymes such as DNase. It is worth noting that high
concentrations of EDTA can also inhibit enzymatic activity during
library preparation and should be reduced as much as possible prior to
library preparation (preferably to < 0.1 mM) (Sambrook et al.,
1989).