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.
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References
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman J (1997) Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Gupta SK, Verma SK, Singh L (2005) Molecular insight into a wildlife crime: the case of a peafowl slaughter. Forensic Sci Int 154:214–217
Gupta SK, Kumar A, Hussain SA (2013) Extraction of PCR-amplifiable DNA from a variety of biological samples with uniform success rate. Conserv Genet Resour 5:215–217
Kocher TD, Thomas WK, Meyer A, Edwards SV, Paabo S, Villablanca FX, Wilson AC (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. PNAS USA 86:6196–6200
Linacre A, Gusmao L, Hecht W, Hellmann AP, Mayr WR, Parson W, Prinz M, Schneider PM, Morling N (2011) ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations. Forensic Sci Int Genet 5:501–505
Naidu A, Fitak RR, Munguia-Vega A, Culver M (2012) Novel primers for complete mitochondrial cytochrome b gene sequencing in mammals. Mol Ecol Resour 12:191–196
Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Tobe SS, Kitchener AC, Linacre AMT (2010) Reconstructing mammalian phylogenies: a detailed comparison of the cytochrome b and cytochrome oxidase subunit I mitochondrial genes. PLoS One 11:e14156
Verma SK, Singh L (2003) Novel universal primers establish identity of enormous number of animal species for forensic application. Mol Ecol Notes 3:28–31
Acknowledgments
This study was funded by the grant-in-aid of the WII. We acknowledge support from the Director, Dean (FWS), FTO and Nodal Officer (Forensic Cell) of the WII.
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Gupta, S.K., Kumar, A. & Hussain, S.A. Novel primers for sequencing of the complete mitochondrial cytochrome b gene of ungulates using non-invasive and degraded biological samples. Conservation Genet Resour 6, 499–501 (2014). https://doi.org/10.1007/s12686-014-0143-6
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DOI: https://doi.org/10.1007/s12686-014-0143-6