Method for identifying biological species

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C536S024310, C536S023100

Reexamination Certificate

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07816078

ABSTRACT:
The present invention provides a method for identifying species and subspecies in a biological sample through the selective amplification of segments of nucleic acid that code a target region of the cytoplasmatic beta-actin protein, which is present in all the organisms concerned. The method comprises DNA extraction from the sample; amplification of divergent segments of the cytoplasmatic beta-actin gene by PCR or an equivalent technique, using primers of regions with high evolutionary conservation between species and subspecies; and identification of the amplified segment by comparison of its size in base pairs with a pre-established standard of sizes and/or identification of the amplified segment by DNA sequencing and comparison of the resulting sequence with the specific sequence of each species or subspecies present on a computer database.

REFERENCES:
patent: 5645994 (1997-07-01), Huang
Lockley et al., “Intron variability in an actin gene can be used to discriminate between chicken and turkey DNA,” Meat Sci 61:163-168, 2002.
Nakajima-Iijima et al., “Molecular structure of the human cytoplasmic β-actin gene: interspecies homology of sequences in the introns,”PNAS 82:6133-6137, 1985.
du Breuil et al., “Quantitation of beta-actin-specific mRNA transcripts using xeno-competitive PCR,” Genome Res 3:57-59, 1993.
Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York, 1989, chap. 6, pp. 30-33, and chap. 14, pp. 5, 10 and 11.
Bellis, C. et al., A molecular genetic approach for forensic animal species identification, Forensic Science International, 134 99-108 (2003).
Kocher T.D. et al., Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers, Proc. National Academy of Science U.S.A., vol. 86 6196-6200 (1989).
Rodriguez, M.A. et al., Qualitative PCR for the detection of chicken and pork adulteration in goose and mule duck foie grass, Journal of the Science of Food and Agriculture 83: 1176-1181 (2003).
NG, S.-Y. et al., Evolution of the Functional Human β-Actin Gene and Its Multi-Pseudogene Family: Conservation of Noncoding Regions and Chromosomal Dispersion of Pseudogenes, Molecular and Cellular Biology vol. 5, No. 10, 2720-2732 (1985).

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