Nucleic Acid Interactions Practicalities

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Nucleic Acid Interactions Practicalities Primer Picking Review Lab#6 Solution 11/22/2018 Chuck Staben

PCR Experiments Basic Experiment Selecting Primers for known DNAs Regions of genes STS/SNP sites from mass sequencing Designing Degenerate Oligonucleotides 11/22/2018 Chuck Staben

PCR Amplify Exponentially A B 11/22/2018 Chuck Staben

Primers Keep Track of Them ~20 bases Specific for target site Efficient amplification of target no competitive sites no internal “foldback” appropriate for experimental conditions Keep Track of Them 11/22/2018 Chuck Staben

Primer Selection Programs Primer3-Whitehead http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi PRIME (GCG) OLIGO http://www.lifescience-software.com/oligo.htm Algorithms Related, Similar 11/22/2018 Chuck Staben

PCR Primer Considerations 3’-complementarity critical Match Potential mismatch Repeat, etc. very bad Self-annealing WWW 11/22/2018 Chuck Staben

Degenerate Oligos PCR related, similar coding region ...YGMWHFIVSSRGAxxxxxxx xxxxxxxxxxxxxxxxYxTVDPx xxxxxxxxCYHNEDM... PCR related, similar coding region 11/22/2018 Chuck Staben

BackTranslate YGMWHFIVSSRGA TyrGlyMetHisPheIleValSerSerArgGlyAla UAYGGNAUGCAYUUYAUHGUNWSNWSNMGNGGNGCN 2 4 2 2 3 96x 18-mer 4224224 4 4 4 216(64,000x) 18-mer 11/22/2018 Chuck Staben

PCR Amplimers DIAGNOSTIC YGMWHFIVSSRGA... YxTVDP... CYHNEDM 11/22/2018 Chuck Staben

Review Genes Proteins DNA sequences Proteins Similarity/Align elements, analysis and finding Proteins alignments, motifs visualize, analyze DNA sequences formats, finding, utilities Proteins RasMol Similarity/Align BLAST, CLUSTAL Motifs, Analyses BLAST,Entrez 11/22/2018 Chuck Staben

Main Programs GCG BLAST Entrez RasMol graphics, frames, map, gap, bestfit, reformatting, translate BLAST blastn, blastp, blastx, tblastn… Entrez RELATIONAL DATABASE RasMol 11/22/2018 Chuck Staben

Lab 6 BLASTN/BLASTX ENTREZ for sequences GAP, BESTFIT CLUSTAL/PILEUP relational to structures and to DNAs GAP, BESTFIT -ran=10 CLUSTAL/PILEUP Analyze via RasMol 11/22/2018 Chuck Staben