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Lecture5_StepsinDesigningDiagnosticMarker



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Steps in Diagnostic Marker Development Use of Genomics for Diagnostics Detection Sequence based or hybridization based methods for identification Sequence divergence Differences between genera Differences between species Differences between pathovars Races isolates Steps in Genomics based Diagnostic Marker Development 1 Identify a target gene amplify and sequence the ITS or anonymous DNA fragment that will be polymorphic species specific pathovar specific or race specific gene 2 Determine detection method sequence gel electrophoresis hybridization Factors in decision Level of specificity needed Throughput Costs Technology required Available information Steps in Genomics based Diagnostic Marker Development Design primers that uniquely amplify the conserved or diverged sequences Confirm primers with ePCR or BLAST computational procedure that is used to identify sequence tagged sites STSs within DNA sequences e PCR looks for potential STSs in DNA sequences by searching for subsequences that closely match the PCR primers and have the correct order orientation and spacing that could represent the PCR primers used to generate known STSs http www ncbi nlm nih gov sutils epcr Perform PCR possibly nested PCR score for gene presence absence or size difference QC could involve sequencing the initial products Ribosomal DNA Diagnostic Markers Obtain rDNA sequences from query genome and nearest relatives CPGR Genbank rDNA databases Ribosomal DNA Primer database http www psb ugent be rRNA primers index html Ribosomal Database http rdp cme msu edu RISSC database Ribosomal Internal Spacer Sequence Collection http miracle umh es rissc Align the sequences using a multiple sequence alignment tool Design primers in the conserved region that flank the ITS Perform PCR Sequence the products Align to query genome and all other sequences available Use of ITS as a diagnostic marker P parasitica Obligate biotroph Diagnosis is delayed on seedlings until sporulation Can develop a rapid



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