**1. Introduction**

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Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. It was established previously that the mitochondrial gene cytochrome *c* oxidase I (COI) can serve as the core of a global bio- identification system for animals. A new tools were developed recently to be complementary markers for (COI) DNA barcoding.

Species identification is essential in food quality control procedures or for the detection and identification of animal material in food samples. Recent food scares e.g. avian flu and swine flu, malpractices of some food producers and religious reasons have tremendously reinforced public awareness regarding the composition of food products. However, because labels do not provide sufficient guarantee about the true contents of a product, it is necessary to identify and/or authenticate the components of processed food, thus protecting both consumers and producers from illegal substitutions [1]. In addition, trade of endangered species has contributed to severe depletion of biodiversity.

Numerous analytical methods that rely on protein analysis have been developed for species identification, such as electrophoresis techniques [2], immunoassays [3] and liquid chromatography [4]. However, these methods are of limited use in species identification. The progress of molecular biology introduced a new approach, which is based on nucleotide sequence diversities among species in particular regions of DNA [5–7]. The nucleotide regions chosen for species identification were varied by researchers. Within vertebrates, a cytochrome b (cyt b) gene in the mitochondrial DNA has been studied from multiple viewpoints including the nucleotide diversity among species [6] and the availability of nucleotide sequence data for references [5]. Many of the other regions studied are also located in the mtDNA. The coding regions for 12S and 16S ribosomal RNA [8–10], and the noncoding D-loop region [7, 11, 12] have shown their potential to be the targets for the species test.

© 2012 Ramadan and Baeshen, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Ramadan and Baeshen, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Although central to much biological research, the identification of species is often difficult. DNA sequencing, with key sequences serving as a pattern ''barcode'', has therefore been proposed as a technology that might expedite species identification [13].

Biological Identifications Through DNA Barcodes 111

IL0500: 5'AGGCATTTTCAGTGCCTTGC-3' IL0501: 5'TAGTGCTAATACCAACGGCC-3'

SH-1: 5' CCT CGC ATG TAC GGC ATA CA-3' SH-2: 5'CAA CCC TTC AGG CAA GGA TC-3'

the 28S.

*the birds* 

*2.2.4. 12S primers* 

*2.2.5. 16S primers* 

Amersham Pharmacia Biotech (U.K.).

Two additional new forward primers (SH-1 and SH-2) specific for buffalo were designed

Two target DNA fragments of the predatory mite, *A. swirskii* were PCR amplified and sequenced: a fragment in the central part of the mitochondrial cytochrome oxidase subunit I gene (COI) and the fragment of the nuclear ribosomal transcribed spacers (ITS) [25-26]. The COI primers were designed specifically for tetranychid mites. They were:

The ITS region was amplified using the primers 5'AGAGGAAGTAAAAGTCGTAACAAG 3' for the 3' end of 18S rDNA and 5' ATATGCTTAAATTCAGGGGG 3' for the 5' end of

*2.2.3. Primers used for amplification of the first 539 base fragment of the D-loop region of* 

The conserved primer pair, L16750 (forward; 5'-AGG ACT ACG GCT TGA AAA GC-3') and H 547 (reverse; 5'- ATG TGC CTG ACC GAG GAA CAA G-3') were used to amplify the first 539 base fragment of the D-loop region of the birds. The primer number refers to the

5'-CAAACTGGGATTAGATACCCCACTAT-3'; 5'-AGGGTGACGGGCGGTGTGT-3' and directed towards the two conserved regions of the gene. The primers were synthesized by

PCR amplification and direct sequencing With two universal primers (sense, 5'- GTGCAAAGGTAGCATAATCA-3' and antisense, 5'-TGTCCTGATCCAACATCGAG-3') directed toward conserved regions **[24]**, the polymerase chain reaction was used to amplify homologous segments of mitochondrial 16S rRNA from four animal species belonging to

5'TGATTTTTTGGTCACCCAGAAG3' and 5'TACAGCTCCTATAGATAAAAC 3'.

inside the D-loop sequence to facilitate sequencing and correction processes.

*2.2.2. Primers used for amplification of specific fragments from mites* 

positions of the 3' end of the primer in the reference sequence **[27].** 

family Bovidae, including river buffalo, cattle, sheep and goat.

Primers specific for mitochondrial 12S rRNA gene were synthesized [23]:

DNA barcoding promises fast, accurate species identifications by focusing analysis on a short standardized segment of the genome [14]. Several studies have now established that sequence diversity in a 650-bp fragment of the mitochondrial gene cytochrome c oxidase I (cox1; also referred to as COI) provides strong species-level resolution for varied animal groups including birds [15], fishes [16] and Lepidoptera [17].

Besides the cox1 gene, other mitochondrial markers also have been widely sequenced across vertebrates for their utility in phylogenetic or to complement cox1 in DNA barcoding.

In amphibians the 16S ribosomal RNA gene (16S) has been suggested as a complementary DNA barcoding marker [18]. Another protein coding gene, cytochrome b, has also been suggested as a marker to determine species boundaries [19, 20].

An attempt was made to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of 'DNA barcoding' [21]. Another study showed that DNA arrays and DNA barcodes are valuable molecular methods for biodiversity monitoring programs [22]. In this chapter we introduce the use of specific fragments of mitochondrial ribosomal RNA from Egyptian buffalo to be used as a perfect barcode for identification of closely related species. Also, we will extend this study to include distantly species identification [23-24]. Our studies were also extended for chickens and small organisms like mites to be studied by both nuclear and mitochondrial markers. Identification of these mites is very important for biological control programs.

All these methods could be used for global bio-identification system or forensic science development.
