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

被引：286

作者：

Deagle B.E. ^{[1
,2
]}

Eveson J.P. ^{[3
]}

Jarman S.N. ^{[2
]}

机构：

[1] School of Zoology, University of Tasmania, Box 252-05, Hobart, Tasmania

[2] Australian Antarctic Division, Kingston, Tasmania, Channel Highway

[3] CSIRO Marine and Atmospheric Research, Box 1538, Hobart, Tasmania

来源：

Frontiers in Zoology | / 3卷 / 1期

关键词：

Faecal Sample; Fragment Size; Faecal Extract; Average Fragment Size; Random Degradation;

D O I：

10.1186/1742-9994-3-11

中图分类号：

学科分类号：

摘要：

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 (λ) 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 λpredator = 0.0106 per nucleotide; mean λprey = 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. © 2006 Deagle et al; licensee BioMed Central Ltd.

引用

共 34 条

[1]

Morin P.A., Chambers K.E., Boesch C., Vigilant L., Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus), Mol Ecol, 10, pp. 1835-1844, (2001)

[2]

Jarman S.N., Gales N.J., Tierney M., Gill P.C., Elliott N.G., A DNA-based method for identification of krill species and its application to analysing the diet of marine vertebrate predators, Mol Ecol, 11, pp. 2679-2690, (2002)

[3]

Withler R.E., Candy J.R., Beacham T.D., Miller K.M., Forensic DNA analysis of Pacific salmonid samples for species and stock identification, Environ Biol Fishes, 69, pp. 275-285, (2004)

[4]

Glenn T.C., Stephan W., Braun M.J., Effects of a population bottleneck on Whooping Crane mitochondrial DNA variation, Conserv Biol, 13, pp. 1097-1107, (1999)

[5]

Shapiro B., Drummond A.J., Rambaut A., Wilson M.C., Matheus P.E., Sher A.V., Pybus O.G., Gilbert M.T.P., Barnes I., Binladen J., Willerslev E., Hansen A.J., Baryshnikov G.F., Burns J.A., Davydov S., Driver J.C., Froese D.G., Harington C.R., Keddie G., Kosintsev P., Kunz M.L., Martin L.D., Stephenson R.O., Storer J., Tedford R., Zimov S., Cooper A., Rise and fall of the Beringian steppe bison, Science, 306, pp. 1561-1565, (2004)

[6]

Lindahl T., Instability and Decay of the Primary Structure of DNA, Nature, 362, pp. 709-715, (1993)

[7]

Paabo S., Ancient DNA - Extraction, Characterization, Molecular-Cloning, and Enzymatic Amplification, Proc Natl Acad Sci USA, 86, pp. 1939-1943, (1989)

[8]

Hoss M., Jaruga P., Zastawny T.H., Dizdaroglu M., Paabo S., DNA damage and DNA sequence retrieval from ancient tissues, Nucleic Acids Res, 24, pp. 1304-1307, (1996)

[9]

Mitchell D., Willerslev E., Hansen A., Damage and repair of ancient DNA, Mutat Res-Fundam Mol Mech Mutagen, 571, pp. 265-276, (2005)

[10]

Ayala-Torres S., Chen Y.M., Svoboda T., Rosenblatt J., Van Houten B., Analysis of gene-specific DNA damage and repair using quantitative polymerase chain reaction, Methods, 22, pp. 135-147, (2000)

← 1 2 3 4 →