Transcript Mfold
Bioinformatics 91-
ucleic Acid Secondari
Structure
AND
Primer Selection
http://gcg.nhri.org.tw:8003/gcg-bin/seqweb.cgi
Nucleic Acid Secondary Structure
Stemloop and Mfold
In Nucleic acids, inverted repeat sequences
may indicate foldback (self pairing)structures.
Identifying Inverted Repeats Stemloop
Calculating RNA Folding
Mfold
Displaying of Folding Structures
Plotfold/Dotplot
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
end
size
217 AGGCTGCAGTG AGCCGTGAT 11, 25
|||||| ||||
C quality
257 TCCGGCCTCAC GTCACCGCG
stem
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.
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
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.
Loop
1
2
3
4
5
6
7
8
217 AGGCTGCAGTG
|||||| ||||
257 TCCGGCCTCAC
AGCCGTGAT
C
GTCACCGCG
135 TAGCCGGGCGT
||| || ||||
160 GTCCGCGCGCG
GG
11, 22
GT
4
Start
217
135
139
69
4
213
221
35
End
257
160
160
95
25
247
248
54
Size
11
11
8
7
9
8
7
8
11, 25
19
Quality
25
22
20
20
20
19
18
18
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
1
35
54
8
18
*.stem
*.pnt DOTPLOT
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
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
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
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
PLOTFOLD
plotC) circles
PLOTFOLD
D) domes
PLOTFOLD
E) mountains
The program plots representative secondary structures that satisfy the energy increment and
window size criteria you specify.
PLOTFOLD
F) squiggles
Exercise 07
StemLoop & Mfold
link
Open the file “Exercixe91-07-1.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 *) ?
Primer Selection
Nucleotide
sequences
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
Specificity - %GC Dimer – Hairpin - Tm
Amino Acid
sequences
Pileup
Pretty
Prettybox
CONSENSUS
Nucleotide
Amino Acid
backtranslate
Primer Selection Program-Prime
Confirm by BLAST
http://gcg.nhri.org.tw:8003/gcg-bin/seqweb.cgi
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
Useful bookmarks for probe and primer design:
http://www.operon.com/oligos/toolkit.php
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 www.operon.com then select DNA Synthesis, then select Oligo Toolkit.
http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi
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).
http://www.ncbi.nlm.nih.gov/BLAST/
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.
RNA folding programs on the Web
mfold version 3.0 by Zuker and Turner at Washington Univ. of St.
Louis
http://mfold2.wustl.edu/~mfold/rna/form1.cgi
SStructView: RNA Secondary Structure Java Applet that
visualizes RNA structures as calculated by mfold
http://smi-web.stanford.edu/projects/helix/pubs/gene-combis96/eg-rnafold.html
RNA secondary structure prediction with GenBee at the
Belozersky Institute, Moscow State University, Russia
http://www.genebee.msu.su/services/rna2_reduced.html
Protein Hydrophobicity Server: Bioinformatics Unit, Weizmann
Institute of Science , Israel
http://bioinformatics.weizmann.ac.il/hydroph/
SAPS - statistical analysis of protein sequences
http://www.isrec.isb-sib.ch/software/SAPS_form.html
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.