Identification of mammal species using species-specific DNA pyrosequencing

doi:10.1016/j.forsciint.2007.01.019

Forensic Science International

Volume 173, Issue 1, 15 November 2007, Pages 16-20

https://doi.org/10.1016/j.forsciint.2007.01.019 Get rights and content

Abstract

In forensic casework it is highly relevant to be able to deduce the species origin of an unknown biological sample. For such a purpose we have designed and developed an assay for species identification based on DNA sequencing of two short mitochondrial DNA amplicons. In short, partial 12S rRNA and partial 16S rRNA fragments (∼100 bp) are amplified by PCR followed by direct sequencing using pyrosequencing technique. Due to properties of the chosen targets, the same PCR conditions and primers were used irrespective of the true species of an unknown sample. A total of 28 different mammals present in the European fauna were sequenced both for the partial 12S rRNA and the partial 16S rRNA sequences for accuracy verification. Together the two sequences showed to have a high divergence factor, discriminating almost all mammals. Furthermore, the human reference nucleotide sequences were always at least nine nucleotides different compared to the other sequenced species both at the partial 12S rRNA and the partial 16S rRNA sequences.

Introduction

DNA analysis for human identification is routinely used to solve crimes, paternity disputes and identifications of human remains. So far DNA analysis has not, however, been used in the same extent within the field of wildlife forensics, although it would be just as useful in cases involving illegal harvesting and poaching of animals and plants.

A number of case reports have recently shown the usefulness of DNA analysis for solving cases including endangered species [1], [2], [3]. However, if the true species affiliation is not known a screening method aiming to deduce the true origin is preferable to use prior to a more refined analysis at an individual level. Such a screening assay should work when no prior information about the origin of the forensic sample is available, i.e. universal primers and PCR conditions are preferable.

Although there are a number of somewhat similar assays available [4], [5], [6], [7] our aim was to design a method for the identification of mammals with the focus on forensic conditions, i.e. degraded and low DNA concentration.

We have chosen to analyze mitochondrial DNA sequences (mtDNA). Human mtDNA is circular and double stranded with 16,569 bp that are completely sequenced [8]. Our choice of mtDNA as target is mainly for two reasons: (1) There are hundreds to thousands of copies of mtDNA in each cell [9] thus providing as much DNA molecules as possible minimizing the risk of failure due to low DNA concentrations with degraded templates. (2) Our assay is designed to amplify a nucleotide sequence with universal primers annealing to conserved sequences. The sequence between the primer sites should, however, be species-specific giving the true species affiliation of the sample.

We have chosen to determine the nucleotide sequence of the amplicons by means of the pyrosequencing technique [10]. This technique uses the fact that pyrophosphate (PPi) is released during DNA synthesis. A cascade of enzymatic reactions makes it possible to detect visible light during the synthesis of DNA. The advantages of pyrosequencing is accuracy, flexibility, parallel processing and that it can be easily automated.

Larger mammals present in the European fauna have been used for validation and to verify the accuracy of the method.

Section snippets

Samples and extraction

Muscle tissue samples were obtained from 74 different animals comprising 28 different species (Table 1). DNA was extracted from approximately 25 mg of tissue using QIAamp DNA Mini Kit (Qiagen). The DNA concentration was determined by UV absorbance at 260 nm on an Ultrospec 2100 pro (Amersham Bioscience). Five human controls consisting of blood donors were additionally analyzed. The mtDNA from the humans was quantified with a SYBR Green based quantitative real-time PCR [11].

Amplification

Sequences within two

Results and discussion

Illegal harvesting and poaching are threats to a wide range of endangered species around the world. For example the third most common illegal product, after gun and arms, is represented by wildlife products [15]. A way to solve such issues is to perform DNA analysis in a similar way that is used for human identification. In this study we present an assay that aims to be used as a screening method for the species identification of a unknown sample prior to the use of a more refined analysis for

Electronic databases

GenBank: http://www.ncbi.nlm.nih.gov/.
EMBL: http://www.ebi.ac.uk/embl/.
Human Mitochondrial Genome Database: http://www.genpat.uu.se/mtDB/.

Acknowledgements

This study was partly supported by The Board for Research and Development, the National Board of Forensic Medicine, Sweden. We also thank friends and colleges contributing with samples, specially The National Veterinary Institute and The Swedish Museum of Natural History.

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Forensic Science International

Abstract

Introduction

Section snippets

Samples and extraction

Amplification

Results and discussion

Electronic databases

Acknowledgements

References (17)

DNA forensics and the poaching of wildlife in Italy: a case study

Forensic Sci. Int.

Application of species-specific polymerase chain reaction in the forensic identification of tiger species

Forensic Sci. Int.

Was elusive carnivore a panther? DNA typing of faeces reveals the mystery

Forensic Sci. Int.

A molecular genetic approach for forensic animal species identification

Forensic Sci. Int.

Cytochrome b gene for species identification of the conservation animals

Forensic Sci. Int.

A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection

Genomics

Real-time DNA sequencing using detection of pyrophosphate release

Anal. Biochem.

DNA N-glycosidases: properties of uracil–DNA glycosidase from Escherichia coli

J. Biol. Chem.

Cited by (87)

Authentication of meat and meat products using molecular assays: A review

Developmental validation of SpeID: A pyrosequencing-based assay for species identification

Mitochondrial ribosomal genes as novel genetic markers for discrimination of closely related species in the Angiostrongylus cantonensis lineage

Busting the myths: DNA typeability after 48 hours of boil

Development of a DNA metabarcoding method for the identification of fifteen mammalian and six poultry species in food

Non-specific peaks generated by animal DNA during human STR analysis: Peak characteristics and a novel analysis method for mixed human/animal samples