**2.3. The amplification reaction**

The amplification reaction used for amplification of the D-loop fragment was also used (with little modifications in temperature cycling) in the other experiments according to the conditions of each experiment.

Biological Identifications Through DNA Barcodes 113

The mean overall, within group and between groups genetic distances were done using the

Our experience in the field of molecular identification or DNA barcoding through a series of published research papers are represented in this section Results with some illustrated figures and tables are represented here but the complete information could be obtained

Shows the Positional entropy plot of the D-loop for the buffalo, and cow sequences The Bayesian phylogenetic trees of cow and buffalo sequences were constructed using MRBAYES software (Figure 2) and Maximum parsimony tree using the Kimura twoparameter model and the closest neighbor interchange method of the MEGA 3.1 software package (Figure 3). Table 1. Shows the Substitution events detected in complete D-loop

**Figure 1.** Positional Entropy Plot of the D loop for the Buffalo, and cow sequences. The X axis is the nucleotide position, while the Y axis is the entropy (lack of information) at that position. The central region is mainly conserved, and the beginning and end regions are highly variable (shaded areas).

through obtaining the complete published papers from the publication section.

sequences from multiple sequence alignments between cows and buffaloes.

MEGA 4.0 software **[12]**.

**3. Results** 

The amplification reaction was carried out in a 25 μl reaction mixture consisting of 1.25 unit Taq polymerase (DyNAzyme), 1X enzyme buffer (1X is 10 mM Tris-HCl, pH 8.8 at 25 0C, 1.5 mM MgCl2, 50 mM KCl and 0.1% Triton X-100) supplied by the manufacture, 1 μM of each forward and reverse primer, 0.2 mM dNTPs and 100 ng of DNA. The reaction mixture was overlaid with sterile mineral oil and was run in an MJ research PTC-100 Thermocycler. The temperature cycling was as follows: 30 cycles of 45 seconds at 94°C; 1 minute at 58°C and 1 minute at 71°C, followed by a final extension at 71°C for 5 minutes. All PCR amplifications included a negative control reaction which lacked template DNA. No product was seen in any negative control. Small quantities of the reaction products (5 μl each) were used for electrophoresis with an appropriate size marker on 1.5% agarose in 1X-Tris acetate buffer (TAE).

After electrophoresis the gels were stained with ethidium bromide and were examined with UV lamp at a wave length 312 nm to verify amplification of the chosen specific fragment. The PCR products were purified using QIAquick PCR purification kit (Qiagen, Inc.) and the resulting purified products were used in the subsequent sequencing reactions. Sequencing was performed on an Applied Biosystems 310 genetic analyzer (Applied Biosystem) using Big Dye terminator cycle sequencing ready reaction mixture according to manufacturer's instructions (Applied Biosystems).
