Dna sequencer genetic analyzer

DNA extraction and PCR amplification of 156srDNA region. DNA was isolated from the selected isolates coded ………………………..according to Sambrook, et al. [7] . The 16srDNA was amplified by polymerase chain reaction (PCR) using primers designed to amplify 1500 bp fragment of the 16srDNA region. The forward primer was 5' AGAGTTTGATCMTGGCTCAG3' and the reverse primer was 5'TACGGYTACCTTGTTACGACTT3'. The PCR mixture consists of 30picomoles of each primer, 10ng of chromosomal DNA, 200 µM dNTPs and 2.5 Units of Taq polymerase in 50 µl of polymerase buffer. The PCR was carried out for 30 cycles in 94°C for 1 min, 55°C for 1 min and 72°C for 2 minutes. After completion, a fraction of the PCR mixture was examined using agarose gel electrophoresis [8] and the remnant was purified using QIAquick PCR purification reagents (Qiagen). DNA sequences were obtained using an 3130 X DNA Sequencer (Genetic Analyzer, Applied Biosystems, Hitachi, Japan), BigDye Terminator Cycle Sequencing (see details below). The PCR product was sequenced using the same PCR primers. Blast program was used to assess the DNA similarities and multiple sequence alignment and molecular phylogeny were performed using BioEdit software [9] . The phylogenetic tree was displayed using the TREEVIEW program [10] .