Transcript Oligo 7 Primer Analysis Software

Oligo 7
Primer Analysis Software
Rules for PCR Primer Selection
and Presentation of the Software
Ⓒ 2010 Molecular Biology Insights, Inc.
Contents
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1. PCR Primer Selection Criteria
- explanation of primer stability calculations
- what makes a good PCR primer
- other factors besides individual primer characteristics influencing PCR reaction
2. Oligo 7 Analyze Features
- the Sequence window
- general information (the Key Info)
- duplex and hairpin formation
- data about primers (Composition and Melting Temperatures)
- false priming sites and homology analysis
- oligonucleotide stability graph (Tm, DG)
- internal stability and its importance
- sequence frequency
- other DNA analysis options: ORF, restriction sites, DNA calculator
3. Search Options
- search for primers and probes
- other searches
- batch processing
4. Oligonucleotide Database
- multiplexing
5. Concluding Remarks
Primer Stability Calculations
An introduction to the nearest neighbor method
One of the most important characteristics of a primer is its the melting temperature and stability of various its regions. The most
accurate Tm calculation provides the nearest neighbor method. The primer Tm formula is quite complex:
Tm (°C) = DH/(DS + R * ln[C]) + 16.6 log [K+]/(1+0.7*[K+]) – 273.15
where DH and DS are the enthalpy and entropy for helix formation, respectively, R is the molar gas constant (1.987 cal/°C * mol),
and C is concentration of the primer/probe (that is always used in several-fold excess than the target DNA), but fortunately
computer does all the work. To understand complexity of duplex stability calculations it is easier to explain it by showing how free
energy (DG, another measure of DNA helix stability), as the formula is simpler:
DG = DH – TDS
(T is the temperature in °K). Although is looks simple enough, it is not so simple if you actually do it. Here’s the example of DG
calculation of a 4-mer TCGA, hybridizing to a longer strand like this:
Now, the DG can be calculated like this surprisingly long formula:
DG (TC) + DG (CG) + DG (GA) + initiation DG for T + initiation DG for A + DG of 3’-dangling end AC + DG of 5’-dangling end GT
The next slide shows the calculation graphically (data for temperature of 37° ).
DG calculation example
Total DG = 1+1+0.3+0.5-1.3-1.3-2.2 = -2.0 kcal/mol
This is a rare case, as usually the dangling ends are stabilizing. This is shown to alert you
that sometimes adding a base may actually decrease melting temperature of a
duplex. The next slide shows an example where adding even 2 bases has no effect
on the Tm.
Here is a screen shot showing the selected primers and probes:
The Forward Primer Tm is 56.7°, while the Reverse Primer Tm is 56.2° (not much different). By adding one
base on each end, and selecting the fragments as Upper and Lower Oligos, we can see that the Upper Oligo Tm
is 56.7° (no change in Tm) while the Lower Oligo’s Tm rose up to 61° (from 56.2°). Without consideration of
the dangling ends, increasing primer length always increases its Tm. More comprehensive analysis data for
those particular oligos you will see on the Key Info window explanation.
What makes a good primer
1.
High priming efficiency
to achieve this, a good primer should be
- of a reasonably high Tm,
- without dimers, especially on their 3’-ends (to prevent self-extension),
- without hairpin stems, especially on their 3’-ends (to prevent self-priming),
- lacking repetitive sequences to ensure quick and correct annealing.
- all primers/probes in one incubation mixture should not form significant 3’dimers between each other.
2.
High specificity
to achieve this, a good primer should be
- long enough to increase specificity,
- unique, especially at its 3’-end, to avoid false priming,
- moderately stable at its 3’-end (as opposed to highly GC-rich) to ensure that a
very short fragment won’t initialize the extension (too low 3’-end stability hurts
the priming efficiency).
Other factors besides individual primer characteristics
influencing PCR reaction
Besides the primer properties listed on the previous page, PCR efficiency also
depends on the template. Regions with high secondary structures and GC
islands amplify poorly. Oligo 7 penalizes primers that would amplify strong
hairpin structures, decreasing overall scores for PCR primer sets.
If you have to make hard to amplify products, select primers with high scores
(usually with higher Tm and GC contents than standard), and adjust
composition of your PCR mix by adding solvents, additives or
reducing/changing salts. Increasing primer concentration above 1 mmol is not
recommended due to unspecific primer/primer extensions (even if the
software does not show any 3’-dimers).
Oligo 7 Analyze Features: the Sequence Window
The Sequence Window is the central window that shows several features of the sequence and selected
primers. When you open the test sequence, provided with the software, you will see this screen:
The sequence file name is in the top left. In the top you see also 3 tables. The first on the left shows the
sequence length, the reading frame for the translation (protein sequence is a colorful string of letters, at the
bottom), the Current Oligo (sequence surrounded by a grey rectangle) length and position, and its Tm. The
second table lists positions of selected primers, oligos (to select, click the square located on the left of the
oligo name), and the PCR product (for this, you need to select 2 Primers). Click the “i” to get more info
about the selected Oligo. The third table lists sequence features (if sequence annotations/features are
present; you may enter them manually from the Edit Menu).
Oligo 7 Analyze Features: the Sequence Window
Below the tables you see the oligonucleotide stability graph (default is Tm). It is a miniaturized Melting Temperature
Graph Window, available from the Analyze Menu. Below the Tm graph there are a series of horizontal lines or rows.
Each row may contain a graphical representation of various features spread out in the entire sequence, such as
primers positions, PCR product and others. In this example, the first filled row is a pattern of red rectangles
symbolizing weak hairpin loops in yet to be selected primers. The green rectangles below the red row represent
palindromes. The last filled row shows the coding sequence as a blue line, as the Feature Table highlights “CDS”.
The bottom part of the window shows the enlarged part of the sequence, that is highlighted in yellow in the Tm
graph (top left of this picture). The cursor position and the Tm of an oligo that would start at the cursor
position is indicated on the right, just above the position numbers scale. Below the scale you see the DNA
sequence, where the positive strand is in red. If you select primers they would appear above and below
the sequence. Translated protein sequence is displayed in various colors. The colors symbolize codon
frequency (black is the most frequent, see the Open Reading Frames window). The rows below and their
colors symbolize identical features, but enlarged, to those displayed just below the Tm graph. More
detailed explanation is on the next slide.
The graph in colored rectangle is expanded in the zoom area
With Oligo you can select up to 4 primers or probes
Explanation of other symbols in the Sequence Window
The Key Info Windows
The Key Info windows
display basic information
about selected primers or
probes. These are the oligos
discussed in the previous
section, where a longer 13mer oligo (lower left) has
the same Tm as its shorter
11-mer version, displayed
above it. The tm is a melting
temperature calculated
without the dangling ends
effect, and this one
increased by almost 7°.
Note that the
complementary oligos of
the same length, shown on
the right, have the same tm
as expected, but different
Tm, especially the 13-mers.
More statistics on those oligos provide the Composition & Tm windows,
as well as the Concentrations window.
The Duplex Formation Windows
The Duplex Formation
windows display the
strongest 3’-duplexes, the
most stable duplex that is
not necessarily at the 3’end, and the strongest
hairpin.
The DG data are given
with consideration of the
dangling ends and
initiation DG values.
The Mixed Oligos
Duplexes window does
not display hairpins.
The Hairpin Formation Windows
The Hairpin Formation windows display
all possible hairpins in a given oligo,
listed from the strongest one to the
weakest.
When a given segment is involved in
many different structures, the real
melting temperature of a particular
hairpin is lower than calculated due
to competition between the
structures.
The Composition & Tm Windows
The Composition & Tm windows display melting temperatures
calculated with different methods widely available, that is the
nearest neighbor method (Td is a normalized Tm in 1M salt
conditions, while the Tm is a variable value, that depends on salt
and DNA concentration set in the Search Parameters window).
The Tm Calculated
with the %GC method
do not depend on the
Search Parameters
settings. Also, the
simplest method of
Tm calculation (2° for
A or T and 4° for C or
G) is given, followed
by the Tm values of
RNA and DNA/RNA
hybrids.
The second table lists
base composition,
and the last the Tm
values in various salts
and formamide
concentrations
calculated with the
nearest neighbor
method.
The False Priming Sites Windows
The False Priming Sites windows display potential
false priming sites of primers. This particular
example shows an old commercially available
universal m13 reverse primer. The site with P.E. of
244 points is the only real false priming site as
confirmed experimentally (Steffens, D.L., Sutter, S.L.,
and Roemer, S.C. (1993) “An Alternate Universal
Forward Primer for Improved Automated DNA
Sequencing of M13”, BioTechniques 15, 580-582.)
The Homology Windows
The Homology windows display homological sites
for probes, and lists the sites from the highest
similarity to the lowest. The 3’-end is irrelevant in
this case, and homology of a probe (or a primer) is
aligned with the actual DNA strand, and not with its
complement, as in the False Priming Sites window.
When using shorter DNA fragments in hybridization
experiments, a homology below 90% is usually not
significant, and won’t produce false signals.
The Selected Oligonucleotides Window
After a search for Primers &
Probes you may open the
Selected Oligonucleotides
Window. In this example a
search for sequencing
consensus primers was
performed in the files listed at
the bottom of the Selected
Oligonucleotides window (to
search this way you need to
click on the “Subsearches” tab
of the Search for Primers &
Probes window, click the
“Consensus Primers” check
box, select the files and start the search.)
When the “Select consensus oligos” box is checked,
as shown on the picture on the right, Oligo 7 selects
a consensus primer when you double-click the row
displaying info about given primer.
The listing of primer positions may be sorted by the score, oligo position or/and any other column of the
window by clicking (or option-clicking) on the column’s title. A pull-down menu in the upper left allows you
to select either Forward or Reverse Primers display. After search for TaqMan sets or Nested primers you
would have even more choices.
The Oligonucleotide Sets Windows
After a search for PCR Primers (top image),
TaqMan Probes (2nd image) or Nested Primers
(two bottom images) the Oligonucleotide Sets
window opens. By clicking on any row in this
window you are selecting a set. Double
clicking causes opening the PCR analysis
window besides that. The windows display
position numbers of the primers and probes,
the score the set received, the PCR product
length and its optimal annealing temperature
for PCR as well as its GC content. The sets can
be re-sorted when you click on the column
titles.
In case of nested primers results, you may
change the display from the external primers
PCR data to the internal by clicking on the
“U/L Product” button, as displayed on the
bottom picture.
More information about any given set is given
in the PCR window, displayed on the next
slide.
The PCR Window
After selecting the primers manually or after a
search for PCR Primers, TaqMan Probes or
Nested Primers you may open the PCR
window. When you have nested primers
selected you may check the external primers
PCR product (“F/R Product”) or nested
primers product data (“U/L Product”).
This window shows graphically the entire
sequence and the location of PCR product and
primers. This window lists the optimal and
maximum annealing temperatures, as well as
the Tm and GC% content of the product and
the primers. A score (quality measure) is also
given. In the table below you see the salt and
primer concentrations, that may be edited at
any time. The comments box displays
warnings when the primers form dimers, have
too low Tm or the product Tm is too high
compared to a primer. In the latter case
expect lower PCR yields than normal but that
itself doesn’t usually completely inhibit the
reaction; the threshold for this warning is
30° Tm difference between the less stable primer and the product. A warning in a set does not disqualify it, but
only suggests that it may be not the most optimal choice. When the warning is “Terminal stability of the Forward
Primer is too high“ it doesn’t mean that the reaction would be impaired at all. It means that when using complex
substrates, such as genomic DNA or reverse-transcribed total mRNA you may have a problem with false product
formation. It would be necessary to carefully check at least the size of the predicted product.
The LCR Window
This window helps designing probes for the ligase chain reaction. When you click on any of the mutation buttons
at the upper left, a special oligo is designed to detect this mutation by LCR. When the enzyme ligates a longer
oligo, with a tail, you would be able to detect it by gel electrophoresis.
The Melting Temperature Graph Windows
The Tm window displays enlarged Tm graph of the Sequence window. It also controls the Sequence Window graph
display. On this example you see that the Tm graph is really the tm graph, as displayed at the top right corner of the
window. This means, that these are “standard” melting temperatures, where the dangling ends are not considered
and therefore the oligos from the both stands have identical Tms. In “real life” this is not the case as the Tms are
different of a given oligonucleotide and its complement because there are different dangling ends (when the target is
a long strand). This window has several options that allow display the Tm graphs with dangling ends as well the DG
values with or without the dangling ends. To change the display, click on the red graph icon at the top left of the
window:
The Melting Temperature Graph Windows, continued
When clicked on the “Dg” row, the free energy graph, without the dangling ends, would be displayed. You may also
choose the GC% contents display or the degeneracy plot, if you have a degenerate sequence file. Note that the Tm , DG
GC% or the degeneracy data are given for the DNA fragments of the Current Oligo size (displayed in red letters and
changeable with the Change-Current Oligo Length command).
At the top of the window there are also zoom-in and zoom-out icons, resembling loupes. You may select different
magnification styles. For example, zooming out creates this type of window:
that resembles more the graph seen in the Sequence Window. Important detail to note is that when you change the
display type in the Melting Temperature Graph Window (this Window may be called differently, depending on the
graph type) you are also changing the display type in the Sequence Window, accordingly.
The “style” icon lets you further modify this window display. The
dot and bar types are available for the graph.
You may also see the average Tm for all the oligos in the file
plotted, and the Search thresholds for the Tm (see the arrows
pointing to those values on the left picture).
The Internal Stability Windows
The windows show DG plots of pentamers, that is, each dot represents the DG of a 5 base-long DNA fragments. A 21mer oligo has 17 pentamers, for example. It is important to be aware of this feature, as they show GC islands, where
hybridization is more likely to initiate. A high stability at the primer 5’-end or in the middle of it is a welcome feature.
It helps anneal it very quickly and accurately. A moderate to slightly high stability at the primer’s 3’-end ensures
efficient priming (very high 3’-end stability may cause false priming though). Internal stability is also important in
other DNA or RNA functions. The siRNA internal stability consensus was discovered using this Oligo software window
(Khvorova, A., Reynolds, A, and Jayasena, S.D. (2003) “Functional siRNA and miRNAs Exhibit Strand Bias”, Cell, 115,
209-216).
The importance of the internal stability analysis is presented on the experiment described on the next slide.
The Importance of Internal Stability Analysis
Two series of primers were designed. One was based on a high 3’-stability primer, H17, and another on a low 3’-end
stability primer, L20. The primers contained only 3’-ends of either H17 or L20, so that H4 sequence was CCGC, H5 was
CCCGC and so on, as shown above. For example, an H8 primer was an 8-mer derived from the high 3’-end stability H17
primer, and L10 was a 10-mer derived from the low 3’-end stability L20 primer. A common reverse 20-mer primer was
used (labeled as C). Three annealing temperatures were used: 40°, 52° and 64°, and the question was what is the
shortest primer that would start the amplification. The template DNA was the wild type bacteriophage lambda.
The Importance of Internal Stability Analysis
PCR results with the low stability 3’-end primer is shown on the left and results with the H-primer series are shown on
the right. It turns out that at 52° annealing temperature (optimal for the LC product; Ta for the HC product is only
slightly higher) you need an 11-mer of the low 3’-end stability primer and only 7-mer of the high 3’-end stability
primer to make a successful PCR. The PCR analysis window for the LC primer pair is in the top right, and the Key Info
windows for all the primers is shown below. The full length primers have very similar Tms, but look at the yields.
The Importance of Internal Stability Analysis, continued
The outcome of the experiment presented on the previous
slide shows that primers with very stable 3’-end are very
efficient, but it’s hard to come by with such primers because
there is a good chance that a false priming or a 3’-end
dimerization may occur. When the 3’-ends of primers overlap, like it’s shown on the Mixed Oligos Duplexes window,
there is a little chance that PCR would be successful. The graph on the top left shows approximate PCR yields and 3’end primer dimer formation dependence. The stronger the dimer the lower the PCR yield. Even more problems may
cause a single primer that forms a 3’-end dimer, because a stable hairpin loop is formed (see the example below).
Designing primers with
not so extremely stable
3’-ends lessen the
chances of harmful 3’-end
dimer formation, and
minimize false priming
probability, so in general,
it is easier to work with
such primers.
The Sequence Frequency Window
When you take a small fragment of DNA, such as 6, 7, or 8-mer, the frequency of hits in genomic databases for such
fragments vastly depends on their sequence. For example, if the average sequence frequency is 1000, the sequence
frequency of TCGCGTA is only 24 and of TGTGTGT is 6789 in Primate DNA library. This means, that when the 3’-end of
any primer is unique (has a low sequence frequency number), there is a low probability of false priming. OLIGO uses
this type of analysis, and if a potential primer has frequent 3’-end its score is lowered or even the potential primer is
eliminated.
The Oligo.net web site provides the software for free, that creates sequence frequency tables from you own
databases, compatible with Oligo software. It creates frequency tables of 6, 7 and 8-mers. The software is called the
Sequence Frequency Calculator, and runs on Windows and Macintosh computers.
The Open Reading Frames Window
This window shows all 6 reading frames of a DNA sequence. The open reading frames are presented graphically in the
upper portion of the window, and as text in the lower part, in a similar manner as the Sequence Window (the double
arrows show the expansion). Click on any of the colored lines or rectangles to see the “ORF Statistics”, that is, the
molecular weight and the logarithmic measure of the acid dissociation constant, pKa of the translated DNA fragment
into protein. In the top left there is a zoom scale that may enlarge the graphics, and next to it there is the “settings”
icon where you may change the amino acid color coding from codon frequency to amino acid type. The color coding is
shown here:
To change the reading frame in this and also in the Sequence Window, simply click on a desired row of amino acids or
on the rectangles or lines (ORFs) in the upper part of the window. Clicking on any base or amino acid symbol changes
position of the Current Oligo as well. The color coding in the Sequence Window depends on this Window settings.
The Restriction Enzyme Sites Windows
You may open this window after a search for the restriction sites. Like the previous window, it also consists of two
parts, graphical (top) and text (bottom). In the graphical section, the entire search area is presented, with a scale bar
at the top. Blue vertical lines represent the restriction enzyme recognition sites. The text section is a table where the
enzyme name, recognition site, number of sites, positions and fragment sized are listed. The enzyme names are listed
alphabetically.
The Restriction Enzyme Sites Windows
This window looks very similar to the previous Analysis window, but it has a totally different functionality. First, you
need to choose the reading frame, than you start the search. OLIGO will translate this reading frame into protein using
degenerate method. Than, it will search all possible DNA combinations for the presence of restriction sites and lists
the results. This does not mean that your original DNA sequence has those sites. It means only that it is possible to
generate a DNA mutation, without changing any coded amino acid, so that any listed restriction site could be created.
The Hybridization Time and Concentrations Windows
With the Hybridization Time Window you may calculate the speed of complementary strands annealing. The T value
displayed in this window is 5 times the T1/2 (50% equilibrium), that is sufficient to reach about 90% equilibrium.
The Concentrations Window is a DNA concentrations calculator. Both windows have editable boxes that you may enter
your own values. When you change the values in the “Constant Concentration” mode you would see changing the
volume, but in the “Constant Volume” mode, changing amount of a given oligo would change its concentration.
Oligo 7 Search Options: the Search for Primers & Probes Window
This is the main OLIGO search window, where you may set up a search for all sorts of primers and probes that are
listed in the Search Options Window. Each type of search require a different set of sub-searches to be performed, that
can be customized in the Subsearches Window. For each subsearch there is a separate set of Parameters, that are
accessible from this Search Window. You may also open the Ranges Window and narrow the search ranges. If you
check the “Continue Above Search in Other File(s)” box the search would be extended on other files thus finding and
eliminating more false priming primers, if necessary. If you check the “Consensus Primers” box, only those primers (or
probes) would be selected from the current sequence file that are common to every additional sequence file that you
choose. The “Apply” button in the Search Options Window sets a new search option and changes all the subsearches
and appropriate search parameters, so that you may check what settings would be used during the search. If you
don’t need to study the settings, than clicking the Search button accomplishes the same thing and also starts the
actual search.
The Search Parameters Window
This window allows you to manually change search parameters, constraints, sequence constraints and even re-define
the scoring system. Every different search type assigns different scores to certain parameters, so that one
oligonucleotide fragment may have a different score, depending if it is a hybridization probe, sequencing primer or a
PCR primer. You may also choose oligos with specific bases at their 3’ or 5’-ends. For example, the default search for a
TaqMan probe avoids G bases at the 5’-ends because they may quench fluorescence. This window has 6 sub-windows.
For a simplicity sake, only two more sub-windows would be shown on the next slide, the Constraints Windows.
The Search Parameters Window, continued
The Constraints Windows allow for manual changing the search parameters. To keep the changed value independent
of automatic stringency changes you need to click on an open lock displayed to the left of each parameter. Clicking
closes the lock. The blue dots next to some locks indicate what parameters would be relevant to the selected search
method.
The Search Ranges Window
The Search Ranges Window lets you choose the search region or regions for all Primer & Probes type searches, PCR
product length ranges and controls whether you want to see the overlapping primers or probes in the results. You may
select several different features to be your search target.
The Search for a String Window
This feature lets you find specific or degenerate sequences. The
results are displayed in the Selected Bases Window in the “Strings”
column and also in the Sequence Window as small orange
rectangles, as indicated by the arrows. You may allow any number of
unspecified mismatches in this search.
The Search in Several Sequence Files: Batch Processing
You may search for primers and probes in several sequence files at
once and save the results in 3 different formats. The button
“Method” opens the standard Search for Primers & Probes Window,
where you may choose the search method. The Parameters and
Ranges Buttons open the same windows as shown on the previous
slides as well. The results can be saved in a simple format text file,
where only the search parameters and oligonucleotide sequences
are listed, or in a text file with full analysis data or as the Oligo
database file that may be opened and analyzed by Oligo at a later
time.
The Oligonucleotide Database
Oligonucleotide Database stores oligos or sets of oligos in a separate document. Besides the main window that lists the
separate entries and sets you may analyze the entries for dimers and hairpins in separate windows. The database has its
own parameters that affect Tm and DG calculations. The P.E. and Tm columns (empty here) would show the real priming
efficiency and Tms if you’d link the database to a sequence of interest and choose Analyze-Priming Efficiency. The p.e. and
tm values are calculated without the supporting sequence and assume perfect matches and no dangling ends. The
database has powerful multiplexing analysis option that is shown on the next slide.
The Oligonucleotide Database: Multiplexing
You may multiplex individual oligos or the entire sets. Here is the result of multiplexing all the sets with the “Minimize
Reaction #” mode. The red numbers represent the set number and the {numbers} represent the database entry numbers.
All oligos in a given group are compatible with each other, that is, they would not form 3’-end dimers that exceed the
search threshold specified in the Database Parameters.
Concluding Remarks
The File-Print/Save Options let you customize the ⌘X (Ctrl-X in Windows OS) short cut key to open any windows you’d
desire. You may also customize the contents of the data file (saved always in text format) of several windows. For example
you may save only plain database records or you may save the full analysis of each record in it.
Oligo Primer Analysis Software was the first software of its kind on the market, released in 1989 (Wojciech Rychlik and
Robert E. Rhoads (1989) A Computer Program for Choosing Optimal Oligonucleotides for Filter Hybridization, Sequencing
and in vitro Amplification of DNA; Nucleic Acids Research 17, 8543-8551), and it has been the leading software of its kind
since then. The current version of Oligo has been described in 2007: Wojciech Rychlik (2007). "OLIGO 7 Primer Analysis
Software". Methods Mol. Biol. 402: 35–60. There are over 1000 citations of this
software, and its number grows all the time. The most recent citations are listed
on Wikipedia (http://en.wikipedia.org/wiki/OLIGO_Primer_Analysis_Software).