1 / 12

Nucleic Acid Interactions Practicalities

Nucleic Acid Interactions Practicalities. Primer Picking Review Lab#6 Solution. PCR Experiments. Basic Experiment Selecting Primers for known DNAs Regions of genes STS/SNP sites from mass sequencing Designing Degenerate Oligonucleotides. Amplify Exponentially. A. B. PCR. Primers.

barr
Télécharger la présentation

Nucleic Acid Interactions Practicalities

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Nucleic Acid InteractionsPracticalities Primer Picking Review Lab#6 Solution Chuck Staben

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

  3. Amplify Exponentially A B PCR Chuck Staben

  4. Primers • ~20 bases • Specific for target site • Efficient amplification of target • no competitive sites • no internal “foldback” • appropriate for experimental conditions Keep Track of Them Chuck Staben

  5. 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 Chuck Staben

  6. PCR Primer Considerations • 3’-complementarity critical • Match • Potential mismatch • Repeat, etc. very bad • Self-annealing WWW Chuck Staben

  7. Degenerate Oligos ...YGMWHFIVSSRGAxxxxxxx xxxxxxxxxxxxxxxxYxTVDPx xxxxxxxxCYHNEDM... PCR related, similar coding region Chuck Staben

  8. BackTranslate YGMWHFIVSSRGA TyrGlyMetHisPheIleValSerSerArgGlyAla UAYGGNAUGCAYUUYAUHGUNWSNWSNMGNGGNGCN 2 4 2 2 3 96x 18-mer 4224224 4 4 4 216(64,000x) 18-mer Chuck Staben

  9. PCR Amplimers YGMWHFIVSSRGA... YxTVDP... CYHNEDM YGMWHFIVSSRGA... YxTVDP... CYHNEDM DIAGNOSTIC Chuck Staben

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

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

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

More Related