Your DNA may be degraded
DNA degradation can result from:
- Using very old DNA samples
- Using DNA extracted from formalin-fixed paraffin embedded samples
- Freezing and thawing DNA samples repeatedly
- Leaving DNA samples at room temperature
- Exposing DNA samples to heat or physical shearing
- Purifying DNA samples inefficiently so residual nuclease remain
Run an agarose gel to determine if the DNA is degraded. Look for a tight band of high molecular weight; smearing indicates degraded DNA.
Agarose gel stained with ethidium bromide showing heat degradation of genomic DNA. This gel shows progressive degradation with increasing time of two human genomic DNA samples subjected to heating at 99°C for 0 to 30 minutes.
As the average size of the DNA in a degraded sample approaches the size of the target sequence, the amount of PCR product generated is reduced. This is due to the reduced number of intact templates in the size range necessary for amplification. An example of degradation by heating is illustrated below. Genomic DNA degraded by other causes will also deliver poor assay results.
Allelic discrimination plots showing the effects of DNA degradation caused by heating.
Factors that affect DNA degradation include tissue preservation methods, exposure to UV radiation, temperature, pH, and salt concentration of the environment (Dean, M. and Ballard, J.W.O., 2001). There are many sources of genomic DNA including fresh capillary blood, buccal scrapes, solid organ biopsies, and paraffin embedded tissue. The table below provides recommended sample storage conditions to help minimize DNA degradation.
Tissue storage conditions
|Tissue Type||Storage Conditions|
|Buccal tissue||Store frozen at –15 to –25˚C|
|Tissue||Immediately place in liquid nitrogen and store at –80˚C, or Freeze and store|
Use more caution in interpreting the results if the DNA is substantially degraded. If possible, consider repeating the assay using freshly prepared genomic DNA samples.