Novel primers for sequencing of the complete mitochondrial cytochrome b gene of ungulates using non-invasive and degrade
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TECHNICAL NOTE
Novel primers for sequencing of the complete mitochondrial cytochrome b gene of ungulates using non-invasive and degraded biological samples Sandeep Kumar Gupta • Ajit Kumar Syed Aniul Hussain
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Received: 12 September 2012 / Accepted: 9 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract We describe a set of novel primers for successful amplification of the complete mitochondrial cytochrome b (cyt b) gene of ungulate species. DNA extracted from non-invasively obtained and decomposed samples is found to be degraded and inappropriate for amplification of the complete gene (more than 1 kb) in single PCR amplification. We developed a series of six ungulate-specific conserved primers for the cyt b gene. These primers, in various combinations, amplify 366–1,266 bp fragments. We also used them in multiplex PCR reaction to assess the maximum possible size of the PCR amplification product. Keywords Mitochondrial cytochrome b gene Non-invasive and decomposed samples PCR amplification Ungulate species Multiplex PCR
Introduction DNA sequencing-based species identification relies on the amount and veracity of the sequence data available at the appropriate database. Identification of species from a sample of unknown origin is based on comparison of DNA sequences obtained from the sample against existing sequences in databases (Verma and Singh 2003; Gupta et al. 2005). A lack of reference sequences from the matching species in the database may lead to high-scoring matches with closely related sequences and produce unreliable results (Naidu et al. 2012). The International Society of Forensic Genetics (ISFG) recommends the use of an inS. K. Gupta (&) A. Kumar S. A. Hussain Wildlife Institute of India, Chandrabani, Dehra Dun 248 001, UK, India e-mail: [email protected]; [email protected]
house and authentic DNA database for species identification for forensic validation (Linacre et al. 2011). Mitochondrial DNA (mtDNA) cytochrome b (cyt b) is an appropriate molecular marker for species identification (Tobe et al. 2010). Hence, large amounts of reference sequence data for the complete cyt b gene are strongly needed for phylogenetic and forensic studies. The use of a conserved primer will be extremely useful for generating a database. The mitochondrial genome is a widely preferred locus for the development of conserved primers (Kocher et al. 1989; Verma and Singh 2003). Such primers have proved to be valuable for species identification in the wildlife forensics (Gupta et al. 2005). Recently, a pair of conserved primer for the complete cyt b gene was developed for mammals (Naidu et al. 2012). Amplifying the complete cyt b gene requires high-quality DNA; however, conservation and forensic geneticists often depend upon non-invasively obtained and forensic (mostly degraded) biological samples. Amplification and analysis of longer fragments (more than 1 kb) from DNA extracted from such samples is challenging. A primer amplifying shorter fragments will be more useful for such studies. We are w
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