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

2. 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

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

4. 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

5. Primer Selection Programs
Primer3-Whitehead
PRIME (GCG)
OLIGO
Algorithms
Related, Similar
11/22/2018
Chuck Staben

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

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

8. BackTranslate YGMWHFIVSSRGA TyrGlyMetHisPheIleValSerSerArgGlyAla
UAYGGNAUGCAYUUYAUHGUNWSNWSNMGNGGNGCN
96x 18-mer
216(64,000x) 18-mer
11/22/2018
Chuck Staben

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

10. 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

11. 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

12. 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