Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 2: MATERIALS & METHODSThe retrieval of nucleotide sequences involved using a corresponding accessionnumber which was available from the literature and entered into a Browse Codeoption available in the WebAngis software or alternatively a Query Search option wasselected which involved entering key terms. The output of the search provided a listof all available sequences corressponding to that query. Appropriate sequences wereselected for further in silico analysis.2.2.1.2 PILEUP Multiple Sequence Alignment (MSA) ProgramFollowing the retrieval of nucleotide or protein sequences, a comparative analysis ofthe sequences was performed of the same biosynthetic gene in different species ofactinobacteria using the PILEUP program. A selection of suitable sequences werealigned to generate a multiple sequence alignment (MSA) by using program defaultvalues.2.2.1.3 PRETTY Consensus Sequence ProgramProceeding the MSA, the PRETTY program was used to determine the regions of thebiosynthetic genes of low variability to establish the location of the consensussequence. MSA were submitted to the PRETTY program and a default setting of80% conservation was extracted. These conserved regions or consensus sequenceswere targeted as sites for the design oligonucleotide primers.2.2.1.4 Database Similarity Of Primer Sequence Using the FASTAProgramFollowing the selection of suitable primers, the FASTA http://www.ebi.ac.uk/fasta3/database search program was used to determine the specificity of these primers andpublished primers. The two criterion used to select suitable primers were that theprimers matched with the gene of interest and were specific for actinobacteria species.2.2.1.5 Calculating Annealing Temperatures of PrimersThe annealing temperature of newly designed primers were calculated usingoligonucleotide calculators which are available as freeware, such aswww.microbiology.adelaide.edu.au/learn/index.htm._____________________________________________________________________55