Bistro-Primer - Tool to design and validate specific PCR primer pairs for phylogenetic analysis

BISTRO-PRIMER TOOL TO DESIGN AND VALIDATE SPECIFIC PCR PRIMER PAIRS FOR PHYLOGENETIC ANALYSIS Praful Aggarwal Marquette University, 2011 Polymerase Chain Reaction is a widely used biological technique which helps in amplifying small quantities of DNA. These amplified DNA copies are then used in several other experiments like DNA sequencing, phylogenetic analysis, etc. PCR primers are short subsequences of nucleotides (basic unit of DNA) that help identify larger regions of the DNA sequence. They help in successfully amplifying the target DNA sequence by identifying complementary regions on the DNA template. Therefore, to successfully perform PCR it is imperative to design good quality primers. PCR can be used for identifying the phylogenetic classification of an organism. For example, in an anaerobic digester, there is a diverse microbial community involved that works to digest the waste material into carbon dioxide and methane. The methane produced can be used in the future as a renewable fuel. To identify the microbes involved, researchers use PCR that uses primer pair(s) targeting some specific group of microbes, on the 16S rRNA region of their sequences. This way they are more likely to amplify DNA from specific microbes only which are present in the target group. This could help in the phylogenetic classification of unknown microbes. In this thesis work a PCR primer pair design and validation software tool has been developed. This tool helps in designing primer pairs that amplify a target region in a specific taxonomic rank (e.g. Genus). It uses a novel scoring function to differentiate between the specific and the not-so specific primer pairs. 16S rRNA sequences for four different genera (Syntrophobacter, Syntrophomonas, Methanosarcina and Streptococcus) were used to develop and test the tool. To the best of my knowledge, primer pair(s) specific for amplifying Syntrophobacter or Syntrophomonas have not yet been published and the results from Bistro-Primer after further validation would be the first specific primer pairs for target amplification of these genera.