1. Nucleic Acid Secondarily Structure AND Primer Selection Bioinformatics 90-07

2. Graphic Display in GCG Configuring Graphics Languages and Devices GIF (Graphics Interchange Format) – GIF87a, GIF89a HPGL (HP Graphics Language) – ColorPro, HP7470, HP7475, HP7550, HP7580, LaserJet3 PNG (Portable Network Graphics) – For WWW Browser PostScript ReGIS Sixel Tektronix Xwindows – Dowload x-win412.exe Program CodonPreference DotPlot Figure Frames FrameSearch /PLOt GapShow GrowTree HelicalWheel Isoelectric MapPlot Moment PepPlot PileUp PAUPDisplay PlasmidMap PlotFold PlotSimilarity PlotStructure PlotTest PrettyBox Prime StatPlot TestCode WordSearch - PLOt

3. Exercise 07-1 Configuring X-windows Download x-win412.exe from ftp://163.25.92.42 Double click x-win412.exe, accept all default settings. Start x-win32 Connect to GCG via TELNET gcg 2% go gcg 3% xwindows Use XWindows graphics with what device: Color Workstation Gray Scale Workstation Monochrome Workstation Please choose one ( * COLORWORKSTATION * ) Plotting Configuration set to: Language: xwindows Device: COLORWORKSTATION Port or Queue: GCG_Graphics gcg 4% plottest GIF & PostScript

4. Nucleic Acid Secondary Structure Stemloop and Mfold In Nucleic acids, inverted repeat sequences may indicate foldback (self pairing)structures. Identifying Inverted Repeats Calculating RNA Folding Displaying of Folding StructuresStemloopPlotfold/Dotplot Mfold

5. STEMLOOP StemLoop finds stems (inverted repeats) within a sequence. You specify the minimum stem length (number of nucleotides in a paired stretch), minimum and maximum loop sizes, and the minimum number of bonds per stem (length of nucleotide sequence between the paired regions). 217 AGGCTGCAGTG AGCCGTGAT 11, 25 |||||| |||| C 257 TCCGGCCTCAC GTCACCGCG start end quality size stem Vertical bars ('|') indicating the base pairs. The associated loop is shown to the right of the stem. If either the stem or loop is too long to be displayed in its entirety on the line, then only that part that fits on the line is shown. The first and last coordinates of the stem are displayed on the left, and the length of the stem (size), the number of bonds in the stem (quality), and the loop size are shown on the right.

6. STEMLOOP Output formats 221 TGCAGTG AGCCGTG 7, 18 ||||||| 248 ACGTCAC CGCGCTA 14 Loop Start End Size Quality 1 35 54 8 18 *.stem *.pnt  DOTPLOT 1) See the stems 2) See the stem coordinates 3) File the stems (*.fld) 4) File the stems as points for DOTPLOT 5) Choose new parameters 6) Get a different sequence Sort stems by: 1) Position 2) Quality 3) Size

7. MFOLD Using energy minimization criteria, any predicted "optimal" secondary structure for an RNA or DNA molecule depends on the model of folding and the specific folding energies used to calculate that structure. Different optimal foldings may be calculated if the folding energies are changed even slightly. Because of uncertainties in the folding model and the folding energies, the "correct" folding may not be the "optimal" folding determined by the program. You may therefore want to view many optimal and suboptimal structures within a few percent of the minimum energy. You can use the variation among these structures to determine which regions of the secondary structure you can predict reliably. For instance, a region of the RNA molecule containing the same helix in most calculated optimal and suboptimal secondary structures may be more reliably predicted than other regions with greater variation. Mfold output file: *.mfold

8. MFOLD How to read *.mfold? Survey of optimal and suboptimal foldings A) sub-optimal energy plot B) p-num plot Sampling of optimal and suboptimal foldings C) circles D) domes E) mountains F) squiggles PLOTFOLD

9. A) sub-optimal energy plot The energy dotplot indicates all of the base pairs involved in all optimal and suboptimal secondary structures within the energy increment you specify. The plot takes the form of a two-dimensional graph where both axes of the graph represent the same RNA sequence. Each point drawn in the graph indicates a base pair between the ribonucleotides whose positions in the sequence are the coordinates of that point on the graph

10. PLOTFOLD B) p-num plot This plot shows the amount of variability in pairing at each position in the sequence in all predicted foldings within the increment of the optimal folding energy you specify

11. PLOTFOLD plotC) circles

12. PLOTFOLD D) domes

13. PLOTFOLD E) mountains The program plots representative secondary structures that satisfy the energy increment and window size criteria you specify.

14. PLOTFOLD F) squiggles

15. Exercise 07-2 Stemloop & X-windows Open the file “exercise07-2.doc” and follow the steps. gcg2 4% fetch gb:d00063 d00063.gb_pl1 gcg2 5% stemloop d00063.gb_pl1 There are 16 stems. Would you like to 1) See the stems 2) See the stem coordinates 3) File the stems 4) File the stems as points for DOTPLOT 5) Choose new parameters 6) Get a different sequence Q)uit? Please choose one (* 1 *): Try 1-4 Sort stems by: 1) Position 2) Quality 3) Size Q)uit Please choose one (* 1 *):

16. Exercise 07-3 Mfold Open the file “Exercixe07-3.doc” and follow the steps. gcg2 4% fetch gb:j02061 J02061.gb_vi gcg2 5% mfold j02061.gb_vi  j02061.mfold $ Mfold (Linear) MFOLD what sequence ? j02061.gb_vi Begin (* 1 *) ? End (* 121 *) ? What should I call the energy matrix output file (* j02061.mfold *) ? Plotfold

17. Primer Selection Specificity - %GC - Dimer – Hairpin - Tm Nucleotide sequences Amino Acid sequences CONSENSUS Pileup Pretty Prettybox Primer Selection Program-Prime Amino AcidNucleotide backtranslate Confirm by BLAST

18. Primer Length Minimum - Maximum - ---------------------------------------------- PCR Product Length Minimum - Maximum - --------------------------------------------- - Maximum number of primers or PCR products in output (range 1 thru 2500) Primer DNA concentration (nM) (range.1 thru 500.0) - Salt concentration (mM) (range.1 thru 500.0) - ---------------------------------------------- Select: forward primers, only reverse primers, only primers on both strands for PCR Set maximum overlap (in base pairs) between predicted PCR products Forward strand primer extension must include position Reverse strand primer extension must include position ---------------------------------------------- Reject duplicate primer binding sites on template Specify primer 3' clamp (using IUB ambiguity codes) ----------------------------------------------- Primer % G+C Minimum (range 0.0 thru 100.0) Maximum ----------------------------------------------- Primer Melting Temperature (degrees Celsius) Minimum (range 0.0 thru 200.0) Maximum ----------------------------------------------- Maximum difference between melting temperatures of two primers in PCR (degrees Celsius) (range 0.0 thru 25.0) ----------------------------------------------- Product % G+C Minimum (range 0.0 thru 100.0) Maximum ----------------------------------------------- Product Melting Temperature (degrees Celsius) Minimum (range 0.0 thru 200.0) Maximum

19. Exercise 07-4 Primer Selection Use the human npm cDNA sequence to design a pair of primers that will copy the whole coding sequence when translated in frame. THEN Check the specificity of the primers by using BLAST.