**2. Materials and methods**

#### **2.1. DNA purification**

Genomic DNA was extracted from peripheral blood of Egyptian buffalo's and chickens by using standard commercial Kit (Pure-gene Genomic DNA purification Kit) as recommended by the manufacturer (www.gentra.com). In case of mites, Genomic DNA was extracted using Capture Column kit method, total DNA was purified using generation DNA purification system**.** 

#### **2.2. Primers used for amplifications of PCR specific fragments**

#### *2.2.1. D-loop primers*

These primers yielded a PCR product of 1142 base pairs. This encompasses the whole of the D-loop and includes flanking sequence at both ends [12].

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

110 Biodiversity Conservation and Utilization in a Diverse World

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

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

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

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

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

Genomic DNA was extracted from peripheral blood of Egyptian buffalo's and chickens by using standard commercial Kit (Pure-gene Genomic DNA purification Kit) as recommended by the manufacturer (www.gentra.com). In case of mites, Genomic DNA was extracted using Capture Column kit method, total DNA was purified using generation DNA

These primers yielded a PCR product of 1142 base pairs. This encompasses the whole of the

**2.2. Primers used for amplifications of PCR specific fragments** 

D-loop and includes flanking sequence at both ends [12].

proposed as a technology that might expedite species identification [13].

groups including birds [15], fishes [16] and Lepidoptera [17].

suggested as a marker to determine species boundaries [19, 20].

programs.

development.

**2. Materials and methods** 

**2.1. DNA purification** 

purification system**.** 

*2.2.1. D-loop primers* 

Two additional new forward primers (SH-1 and SH-2) specific for buffalo were designed inside the D-loop sequence to facilitate sequencing and correction processes.

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

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

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:

#### 5'TGATTTTTTGGTCACCCAGAAG3' and 5'TACAGCTCCTATAGATAAAAC 3'.

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 the 28S.
