1. Nucleic Acid Secondarily
Bioinformatics 92-05
Nucleic Acid Secondarily
Structure
AND
Primer Selection

3. 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 Structures
Stemloop
Plotfold/Dotplot
Mfold

4. 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).
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.
start
size
217 AGGCTGCAGTG AGCCGTGAT 11, 25
|||||| |||| C
257 TCCGGCCTCAC GTCACCGCG
quality
end
stem

5. StemLoop searches for inverted repeats in your sequence after you choose a minimum stem length and minimum and maximum loop sizes.
You must also specify a minimum number of bonds per stem with G-T, A-T/U, and G-C scored as 1, 2, and 3 bonds, respectively. The stems found can be sorted by position, size (stem length), or quality (number of bonds) and can be either filed or displayed on the screen.
StemLoop tells you the number of stems found for your settings of
minimum stem size,
maximum loop size,
minimum loop size, and
minimum bonds per stem.
If you feel there are too many stems, you may reset the parameters without reviewing the stems found or view only the best stems found. To view only the best stems, there must be more than 25 stems found and you must sort them by quality or size.

6. PARAMETER REFERENCE – STEM LOOP
Minimum stem length (window) sets the minimum stem length. This value cannot exceed either 50 or half the sequence length.
Minimum bonds per stem (stringency) sets the minimum bonds per stem.
Minimum loop size sets the minimum loop size.
Maximum loop size (distance to furthest inverted repeat) sets the maximum loop size (distance to furthest inverted repeat).
Sort stems by:
position quality size indicates how to sort the stems in the output.
Number of stems to show sets the maximum number of stems to show (only applies when stems are sorted by quality or size).
Threshold for nibbling, match (|), and point display
The output from this program has a '|' (vertical bar) between sequence symbols that match. This match display character is added to the output whenever the symbol comparison value for the two symbols in your scoring matrix is greater than or equal to the average positive non-identical comparison value in the matrix. The Threshold for nibbling, match (|), and point display parameter lets you specify a match display threshold appropriate for the scoring matrix you are using.

7. STEM LOOP output file Vertical bars ('|') indicating the base pairs.
The associated loop is shown to the right of the stem.
The first and last coordinates of the stem are displayed on the left.
The length of the stem (size),
the number of bonds in the stem (quality),
and the loop size are shown on the right.
217 AGGCTGCAGTG AGCCGTGAT 11, 25
|||||| |||| C
257 TCCGGCCTCAC GTCACCGCG 19
135 TAGCCGGGCGT GG 11, 22
||| || ||||
160 GTCCGCGCGCG GT 4
Loop Start End Size Quality

8. STEMLOOP Output formats 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
221 TGCAGTG AGCCGTG 7, 18
|||||||
248 ACGTCAC CGCGCTA 14
Loop Start End Size Quality
*.stem
*.pnt  DOTPLOT

9. MFOLD Mfold output file: *.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

10. 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

11. A) sub-optimal energy plot
PLOTFOLD
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

12. 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

13. PLOTFOLD
plotC) circles

14. PLOTFOLD
D) domes

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

16. PLOTFOLD
F) squiggles

17. Exercise 07 StemLoop & Mfold
link
Open the file “Exercixe doc” and follow the steps.
gcg2 4% fetch gb:d00063 and gb:j02061
j02061.gb_vi d00063.gb_pl
gcg2 5%
mfold d00063.gb_pl  d00063.mfold
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 *) ?

18. Primer Selection Program-Prime
Specificity - %GC -
Dimer – Hairpin - Tm
Nucleotide
sequences
Amino Acid
CONSENSUS
Pileup
Pretty
Prettybox
Primer Selection Program-Prime
backtranslate
Confirm by BLAST
Targets an amplicon length of 75 to 150 bp
50 to 60% GC content
Limit secondary structure
Limit stretch of G or C’s longer than 3 bases
No stable interaction between forward and reverse
primers (primer/dimer pairs)
Place C’s and G’s on ends of primers,
but no more than 2 in the last 5 bases on 3’ end
Melting Temperature (Tm) above 50 oC
Verify specificity

21. ----------------------------------------------PCR Product Length
Primer Length
Minimum -
Maximum -
PCR Product Length
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 difference between melting temperatures of two
primers in PCR (degrees Celsius) (range 0.0 thru 25.0)
Product % G+C
Product Melting Temperature (degrees Celsius)

22. Useful bookmarks for probe and primer design:
 Use free online Tm calculators to see what the Tm for primer and probe sequences are. We use the Operon calculator, as it also has a good tool to check possible primer-dimerization sequences. Reach it from then select DNA Synthesis, then select Oligo Toolkit.
 This is a link to Primer3 software. It is software that allows for primer design and also helps picking an internal oligo sequence to these primers (a probe sequence). Like most primer design algorithms, it has the disadvantage of not taking into account secondary structure issues that are paramount in primer/probe design for real-time PCR. With that caveat in mind, it is a good place to start the design process, if you are not inclined to do it by eye (i.e. scanning the sequence yourself). Then you can check your sequences at the folding site (described below).
 This is the site for the Basic Local Alignment Search Tool from the National Center for Biotechnology Information. Use this site for checking specificity of probe and primer sequences.

23. RNA folding programs on the Web
mfold version 3.0 by Zuker and Turner at Washington Univ. of St. Louis
SStructView: RNA Secondary Structure Java Applet that visualizes RNA structures as calculated by mfold
RNA secondary structure prediction with GenBee at the Belozersky Institute, Moscow State University, Russia
Protein Hydrophobicity Server: Bioinformatics Unit, Weizmann Institute of Science , Israel
SAPS - statistical analysis of protein sequences

24. Exercise 05 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.