How to use PrimerExplorer V5

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1. Primer design using M13 as the template (Target)
1.1 Uploading the target sequence
The target sequence is uploaded in the PrimerExplorer V5 startup window (Figure 1.1).
First, click on the “Browse” button to select the target sequence file. The target sequence entered is set to less than
2 k bp. Three types of file formats are supported, plain text format (sequence only), FASTA format, and GenBank
format.
Next, a parameter set (primer design conditions) is chosen from one of the three below.
1) Automatic Judgment: Based on the GC content of the target sequence, the initial parameter setting is specified. If
the GC content is 45% or less, the “AT rich” parameters are used; if greater than 60%, the “GC rich” parameters areused. For all others, the ”Normal” parameters are used.
2) Normal: The user enters the primer design conditions manually to design the primers. As the default conditions,the “Normal” parameters from 1) above are displayed.
3) User Assignment: Click on the [Browse…] button on the right and specify the parameter file of primer design
conditions saved on the PC. The specified parameter file will be used as the initial setting to design the primers.

1) Select the target
sequence file.

2) Specify the

Browse…

parameter set.

Browse…

3) Click on the “Primer
Figure1.1 PrimerExplorer V5 startup window

Design“ button.

The default parameter set is “automatic judgment.” In “automatic judgment,” the GC content of the target sequence is
automatically calculated, and the primer design conditions are automatically selected in the following primer design
conditions (“Normal sequences primer design conditions“ “GC rich sequences primer design conditions,“ “AT richsequences primer design conditions “).
Next, click on the “Primer Design“ button.

111.2 Designing the primer (Easy Mode)
As an example, a portion of the M13 sequence (length 1969 bp, GC content = 48.2%) will be used to design theprimers. Click on the “Generate” button (Figure 1.2). This mode eliminates the need to change parameters anddisplays five primer sets that are likely to have high amplification efficiency. It automatically narrows down andprioritizes primer set candidates. The “Generate sets” box shows that five primer sets have been designed. Clicking
on the “Display” button will display the Primer Set List results (Figure 1.3).

Click on the“Generate” button

Click on the“Display” button

Figure1.2 Primer design window in Easy Mode
A list of primer sets designed on the target sequence appears in a separate window, and you can save the list in an
Excel file by clicking the “Save List” button. Here, check the button at the left end of the window, and click on the“Confirm.” The Primer Set Details window appears with detailed primer information for the checked primer sets
(Figure 1.4). Check for problematic findings in each parameter. Click on the “Primer Information” for each primer set
to save the information for that primer. This information should be used to design loop primers. In this window,
clicking the “Save” button allows the sequence information of each primer to be set in an Excel file.

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“Confirm” button to check thedetails of the primer.

“Save List” button to save theview of the list in Excel format.

Figure 1.3 Primer Set List window

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To save the primer
sequence information,
click on the “Save”

To save the primer information
for use in designing the loop
primers, click on the “Primer
Information” button.

Figure 1.4 Primer Set Details window

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1.3 Designing the primer (Expert Mode)
Although Easy Mode enables you to design primers with some capability, Expert Mode allows you to design with
better capability or customized primers. To jump to the Expert Mode (Figure 1.6), click on the “Detail Setting” button in
Easy Mode window (Figure 1.5). The default parameter set is “automatic judgment”. In “automatic judgment”, the GC
content of the target sequence is automatically calculated, and the primer design conditions are automaticallyselected in the following primer design conditions (“Normal sequences primer design conditions”, “GC richsequences primer design conditions” and “AT rich sequences primer design conditions”). As indicated in the primer
design window, “Parameter Set” of “Normal” has been selected. Normal parameter conditions are as indicated in
Figure 1.6.
Next, click on the “Generate” button to start the primer design. When the primer design starts, the message area will
indicate the status of progress in the primer design. The number of primer candidates for each region that fulfills theparameter conditions is displayed, as well as the number of inner primers (FIP, BIP) for each region. Based on these
data, the primer sets are created. In this example, a total of 1,000 primer sets ware designed (Figure 1.7). Clicking on
the “Display” button will display the Primer Set List result.

Click on the“Detail Settings”
button.

Figure 1.5 Primer design window

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Click on the“Generate” button.

Click on the“Display” button.

“Parameter Set”
of “Normal” hasbeen selected.

Figure 1.6 Expert Mode

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The message area
indicates the status ofprogress.
A total of 1,000 primer
sets were generated.

Figure 1.7 Primer design window
Click on the “Display” button
to display the results.

1.4 Displaying the results
Primer Set List window (Figures 1.5a, 1.5b) shows the ID number of each primer set on the left, and to its right thechange in free energy, which indicates the propensity for dimer formation. A low value of the change in free energy
results in a higher likelihood of dimer formation and thus the primer set is unacceptable. Green capital letters indicate
the region F3, blue capital letters indicate the region F2, black lower-case letters indicate the region F1c, black capitalletters indicate the region B1c, blue lower-case letters the region B2, and green lower-case letters the B3 region.
The primer set is designed with the 5’ end of F2 as the origin, and primer sets that fulfill the primer design
conditions are displayed for the entire target sequence from the 5’ end toward the 3’ end. For each region F2, ones
from other regions (regions F3, F1c, B1c, B2 and B3) are determined and displayed. After displaying the primers
designed for the target sequence from the 5’ end to the 3’ end, the design is re-started from the 5’ end to the 3’ end.
This operation is repeated until 1,000 primer design candidates are generated.
In this example, the length of the input target is 1,969 bp, and after the first round from the 5’ end to the 3’ end, 55
primer sets have been designed. After the second round, primers are designed from set 56 to set 110. The 5’ end of
the region F2 included in the final primer set after the first round is at 1,281 bp (the 5’ end of F3 is 1,439 bp). (SeeFigure 1.5b) Several primers are then selected to compare the specific conditions.

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ID number

Free energy value

Region F3

Region F2

2) Click on the“Confirm” button.

1) Check the box
to the left of theprimer set selected.

Figure 1.8a Primer Set List window-1 (page 1)

Region B2

Region B1c

Figure 1.8b Primer Set List window-2 (page 2)

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Region B3

The 5’ end of B3 is1440bp.

Figure 1.9 Primer Set List (full view)

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1.5 Primer set selection
Three to five or more primer sets that amplify different regions in the target sequence are designed, and theiractual reactivities are compared to select the appropriate primer sets. If the region to amplify is pre-determined, theprimer sets that amplify the region are selected.
If the appropriate primer is selected from among the multiple primer sets designed in the same region, detail
information is compared. Here is an example of comparing two primer sets (ID number 1 and 2) in the Primer Set List
window (Figure 1. 8a). First, check the boxes located to the left of each primer set, and click on the “Details” to open
the Primer Set Details window (Figure 1.10).
Primer length

5’ end stability

Tm

Location of the 3’ end

3’ end stability

Location of the 5’ end

GC content

Sequences

ID number

Figure 1.10 Primer Set Details window

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In the window displayed as in Figure 1.10, check for the stability of the 3’ end at region F2, the 5’ end at region F1c,the 3’ end at region B2, and the 5’ end at region B1c, in each primer set. Since these are the starting points for gene
amplification by primers, their end stability is important. Specifically, check to see whether the ⊿G (stability) is −4.0
kcal/mol or lower. For example, the end with ⊿G = −6.5 kcal/mol is more stable than the end with ⊿G = −4.0
kcal/mol.
There is a “Primer Information” button above each ID number (Figure 1.11). This button should be used to design
loop primers for the primer set selected. To save the primer sequence information, click on the “Save” (Figure 1.11).
Click on the “Primer Information” to save the primer information to be used to explain loop primer design.
To save the primer
sequence information,
click on the “Save”

To save the primer information
for use in designing the loop
primers, click on the “Primer
Information” button.

Figure 1.11 Selected Primer Set

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2. Primer design for AT-rich sequences
In this section, primers will be designed for an AT rich gene sequence. We will use a portion of a viral gene of 1,140
bp in length and GC content = 34.5%.
Upload the target sequence in the Startup window of the PrimerExplorer.
Enter the target sequence file, and after confirming that “Automatic Judgement“ has been selected for theparameter set, click on the “Primer Design“ button. Click on the “Detail Settings” button to change to the Expert
Mode (Figure not shown).

Click on the“Generate”“AT rich” has been
selected for the“Parameter Set”.

The setting includes alonger primer lengthand lower Tm.

Figure 2.1 Primer Design window

The GC content of the sequence was automatically calculated, and the sequence was determined to be AT rich.
Thus, “AT rich” was automatically selected as the “Parameter Set.” The setting calls for a longer primer and a lower Tmvalue (see Figure 2.1).
Next, click on the “Generate” to design the primers. This will result in the design of 1,000 primer candidates (notillustrated). Subsequently, click on the “Display” to display the results of the primer design.
At this point, 147 primer sets have been designed from the 5’ end to the 3’ end. Additional primer sets from the 148th
set have been designed again from the 5’ end to the 3’ end (see Figure 2.2).
The method described in Chapter 1 (see pp. 18-23) is then followed to compare the primer information, and to selectthe primer sets. It should be confirmed that at each primer region, the differences in the Tm between F1 and F2 andbetween B1 and B2 are about 5°C.

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147 sets of primers have been designed from the 5’ end to
the 3’ end and additional sets from the 148th set have been
designed again from the 5’ end to the 3’ end.

Figure 2.2 Primer Set List window< Note>For GC rich sequences, the parameter set for GC rich sequences are automatically selected and theprimers are designed to cover the entire target sequence.

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3. Changing the primer design conditions (parameter)(Precautions in primer design)
3.1 When too many primer sets are generateda) Adjust the primer GC content.
When the primer GC content is 50 - 60%, favorable amplification performance will be obtained experimentally.
Thus, the conditions are adjusted so that the GC content is in this range. Narrowing the permitted range for the GC
content will be able to reduce the number of candidates.
b) The differences in the Tm are set to about 5°C for the primers (regions F2 and F1c, regions B2 and B1c).
In the LAMP reaction process, F1 (B1) and F1c (B1c) each self-anneal to form a loop structure, which serves as
the starting structure for amplification. To facilitate forming this loop, set F1c (B1c) at a Tm value around 5°C higher
than those of the other primers. When less stringent conditions (wider range of Tm’s at each primer location) areused to design the primers, primer sets are generated, which consists of the primers with various Tm value. For thisreason, the difference in the Tm in each primer region may be 3°C or less. Also, best results are obtained if the Tm's
match between regions F2 and B2, regions F1c and B1c, and regions F3 and B3.

3.2 When too few primer sets are generated
If only small number of primer sets is generated for GC rich or AT rich sequences, it is plausible that the primer
design conditions for the given target sequence are too stringent. In PrimerExplorer V5, the primer design conditions
are automatically selected for GC rich or AT rich sequences, but for some sequences, in spite of these conditions
only a few primer sets are generated. In such cases, the range of primer length or the range of Tm should be
adjusted.
a)

For AT rich sequences
For AT rich sequences, the Tm is calculated to be lower than non-AT rich sequences of the same length. For thisreason the Tm based on the default primer length may be lower than the lower limit of default Tm value, and prevent
primers from being designed. Thus, the primer length should be increased and/ or the Tm should be decreased.
b)

For GC rich sequences
In contrast, for GC rich sequences, the Tm is calculated to be higher than non-GC rich sequences of the samelength. For this reason the Tm calculated from the default primer length may be higher than the default Tm upper limit,
and prevent primers from being designed. Thus, the primer length should be decreased and/or the Tm should be
increased. Because how the Tm or the length is adjusted would be determined on a case-by-case basis, the length ofeach primer should be changed by one base at a time and the Tm should be changed 1°C at a time. Once a large
number of primers have been generated, then stop the adjustment and select the primers.

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3.3 Changing and storing the primer design conditions
When designing the primers, the user can change primer design conditions. The primer design conditions can besaved and revised. In this example (Figure 3.1), the Length, Tm, and GC content (%) have been changed. To savethe primer design conditions, click on the “Save Parameters” button. As indicated in Figure 3.2, the program will ask
how the conditions should be saved. Save the primer design conditions by specifying the file name and location.

Click on the “Save
Parameters” button.
Red indicated theLength, Tm, andGC content (%) thatwere changed.

Figure 3.1 Changing the primer design conditions (Primer Design window)

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3.4 Using the saved primer design conditions for the primer designing
Upload the target sequence in the startup window of the PrimerExplorer. Next, check on “User Assignment” in theparameter set and click on the “Browse” button to select the parameter file containing the primer design conditions.
Click on the “Primer Design“ button.

2) Check on the “UserAssignment” and click on

1) Select the target

the “Browse” button.

sequence file

Select the parametercontaining the saved
primer design conditions3) Click on the “Primer

Browse…

Design” button.

Browse…

Figure 3.2 PrimerExplorer Ver. 5 startup window
The primer design window (Figure 3.4) will display the previously saved (Figure 3.2) primer design conditions.
Here, the “Parameter Set” is displayed as “Custom.”Next, click on the “Generate” button to design the primers. The primers are selected using the procedures
described in Section 1 (see p.13 – 18).

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“Custom” has been
selected as the“Parameter Set”.

Previously saved
primer design
conditions will be
displayed.

Figure 3.4 Primer design window
Even if a “User specified” parameter of “Custom” has been selected, it is possible to switch to other primer design
conditions (Normal, AT rich, GC rich). To do this, select another desired primer design conditions in the pull-downmenu in “Parameter Set” prior to designing the primers. (See Figure 3.5)

Select another primer
design conditions in
the pull-down menu.

Figure 3.5 Changing the parameters27
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4. Designing primers with specified primer locations
4.1 Specifying the primer locations in the target sequence
Primer can be designed for a specified primer location if the region is known to be easily amplified by PCR, or if theregion to be amplified is pre-determined, or if it is desired to use the primers or primer locations used in PCR.
As in Figure 4.1, specify the primer location by clicking on the “primer location“ button. The Figure shows that the“F2” button is clicked, and as in Figure 4.2, the region specified as the location F2 will be displayed.
1) Specify theprimer location.

2) Click on this button to
specify the location F2

Figure 4.1 Primer design window

Display of thespecified primer
location

2) Click on the
button to1) To change the specified

re-specify theregion, specify a newlocation F2.

location F2.
Figure 4.2 Window after specifying the primer location

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To change the location F2 to some other location, specify another location as shown in Figure 4.2, and click on the“F2” button again. As shown in Figure 4.3, the new location is now specified as the location F2.
The locations can be changed as above. To delete the information at this primer location, click on the “Clear”
button to delete.

The previously specified location is deleted
and the newly specified location is displayed.

Click on this button
to delete the informationon this primer location.

Figure 4.3 Primer design window

4.2 Specify the primer location to be designated for primer design
Now we design primers in which the primer location has been pre-specified. Here, as indicated in Figure 4.4, thelocation F3 has been pre-specified prior to the primer design. Specify the primer location by clicking on the “F3”button, and once the specified location has been displayed, then click on the “Generate” button to design the primers.
(See Figures 4.4, 4.5)

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111) Specify theprimer location.

2) Click on thisbutton to specify
the location F3

Click on the“Generate” button.

Click on the“Display” button.
Figure 4.4 Primer design window
Primer sets with theF3 specified havebeen designed.

Figure 4.5 Primer Set List window

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5. Loop primer design
5.1 Uploading the primer information file
Return to the PrimerExplorer startup window and re-load the previously saved “primer information file“.

Click onthe “Browse” button to select the file, and then click on the “Primer Design“ button. (See Figure 5.1)

1) Select a primer
information file.

2)

Browse…

Click on the “Primer

Design“ button.

Browse…

Figure 5.1 PrimerExplorer V5 startup window

5.2 Designing loop primers
After uploading the primer data file, the loop primer design window will be displayed as shown in Figure 5.2 on thenext page. Keep the parameters as default and click on the “Generate” button.

Click on the“Generate” button
without changing theparameters.

Figure 5.2 Loop primer design window

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A total of 24 sets of primer will be generated. Click on the “Display” button to display the Primer Set List (See Figure
5.3)2) Click on the

Saved primer“Confirm” button.

information locations

1) Check theboxes.

Forward-side loop
primer

Backward-side loop
primerFigure 5.3 Loop primer design window (after primer design)
Figure 5.3 shows the results as a Primer Set List. At the top is the location of the saved primer information,
underneath is the target sequence, and at the bottom are the loop primers. To examine the detailed information
regarding these loop primer sets, check the boxes to the left of primer sets, then click on the “Confirm” to open thePrimer Set Details window.

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5.3 Detailed information on the loop primer sets
The Primer Set Details window (Figure 5.4) shows the detailed information regarding the loop primer sets
previously selected.

Figure 5.4 Primer Set Details window
After more than one loop primer set has been designed, follow the method described in Chapter 1 (see pp. 17-27)
to select a primer set.

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