Quantification of damage in DNA recovered from highly degraded samples--a case study on DNA in faeces

Abstract

Background: Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (lambda) can be estimated by determining the rate of decline.

Results: The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean lambda(predator) = 0.0106 per nucleotide; mean lambda(prey) = 0.0176 per nucleotide). This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples.

Conclusion: We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will allow researchers to measure template quality in order to evaluate alternate sources of DNA, different methods of sample preservation and different DNA extraction protocols. The technique could also be applied to study the process of DNA decay.

Figure 1

Theoretical proportion of amplifiable fragments versus amplicon size after a random degradation process. Results are shown for cases in which the probability of a nucleotide being damaged (λ) is: 0.00125, 0.0025, 0.005, 0.01 or 0.02.

Figure 2

Overview of the approach for quantification of DNA damage. (a) Primers are designed that amplify fragments of several sizes. The schematic representation shows the position of oligonucleotides and the picture below shows the corresponding PCR products (amplified from genomic DNA) separated on a 1.8% agarose gel. (b) The various sized fragments are amplified using real-time PCR. This representative plot of fluorescence observations shows amplification of herring DNA from a single sea lion faecal DNA extraction. PCR fragment sizes from left to right are 69 bp, 123 bp, 184 bp, 226 bp and 304 bp. (c) Copy number (Ax) estimates are obtained for each of the amplicon sizes (x). The plot shows the amount of herring DNA in a sea lion faecal sample (#7). (d) The data is then log-transformed and a linear model is fitted in order to estimate the probability of a nucleotide being damaged (λ).

Figure 3

Quantitative PCR results and estimates of DNA damage in faecal DNA. Shown is the number of amplifiable copies (logarithmic scale) verus amplicon size for sea lion DNA (blue) and herring DNA (red) extracted from ten sea lion faecal samples. The estimated probability of a nucleotide being damaged (λ) is also shown for each target species in each sample.

Figure 4

Quantitative estimates of the amount of amplifiable herring DNA in three spiked faecal DNA extractions. Horizontal lines show the actual amount of herring DNA added as template (either 3380 or 13520 copies of the plasmid control). Symbols represent the corresponding estimates of the amount of herring DNA in three samples measured with assays targeting PCR products of five different sizes (69 bp, 123 bp, 184 bp, 226 bp and 304 bp).

Figure 5

Plots showing estimated proportion of amplifiable fragments versus amplicon size for various DNA extracts. The predator and prey faecal DNA curves use the mean λ values determined in the current study for sea lion DNA (λ = 0.0106) and herring DNA (λ = 0.0176). The ancient faecal DNA λ was estimated from published data [17] on the quantity of sloth mtDNA in a late Pleistocene sloth coprolite (λ = 0.033). The mammoth DNA λ (0.007) was also estimated from published data [26].