How to use PrimerExplorer V4_1. Primer design using M13 as the template (Target)
1.1 Uploading the target sequence
The target sequence is uploaded in the PrimerExplorer V4 startup window (Figure 1.1).
First, click on the “Browse” button to select the target sequence file.
than 2 kbp.

The target sequence entered is set to lessThree types of file formats are supported, plain text format (sequence only), FASTA format, andGenBank 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 are used.

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 theparameter set.

3) Click on the “Primer
Design“ button.

Figure1.1 PrimerExplorer V3 startup window

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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.

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”
buttonClick on the“Display” button

Figure1.2 Primer design window in Easy Mode

A list of primer sets designed on the target sequence is then shown in a separate window, and you can save that listin Excel format by clicking on the “Save List” button. Here, check the boxes for the primer sets you want to confirm in
the left side of the window, and then click on the “Confirm” button. Another window then appears with detailed primer
information of the checked sets (Figure 1.4). In this window, click on the “Primer Information” button for each primer
set to save the information for that primer. The information should be used to design loop primer.

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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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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” buttonin Easy Mode window (Figure 1.5). The default parameter set is “automatic judgment”. In “automatic judgment”, theGC 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
the parameter conditions is displayed, as well as the number of inner primers (FIP, BIP) for each region. Based onthese 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.

Click on the “Display” button
to display the results.

Figure 1.7 Primer design window

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 capital letters indicate the region B1c, blue lower-case letters the region B2, and green lower-case letters theB3 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’ endof the region F2 included in the final primer set after the first round is at 1,281 bp (the 5’ end of F3 is 1439 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

Region F1c

Click on the“Confirm” button.

Check the box to theleft of the primer set
selected.

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

Region B1c

Region B2

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

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Region B3

The 5’ end of B3 is1,440 bp.

Figure 1.9 Primer Set List (full view)191.5 Primer set selection
10-15 primer sets that amplify different regions in the target sequence are selected, and detail information on thesets is compared to select the appropriate primer sets. If the region to be amplified is pre-determined, then theprimer sets that amplify that region are selected.
Here, we assume that “any region in the target sequence can be amplified.”
In the Primer Set List window (Figure 1.8a), primer sets are chosen that encompasses the entire length if possible.
As an example, the ten sets, with the ID numbers 1, 5, 8, 12, 14, 15, 24, 30, 37 and 40, are selected.

Check theboxes located to the left of each primer set and click on the “Details” button to open the Primer Set Details window.
5’ end stabilityTm

3’ end stability

Primer length
Location of the 3’ end

GC content

Location of the 5’ end

Sequences

ID Number

Figure 1.10 Primer Set Details window

In the window shown in Figure 1.10, check the stability of the 3’ end at the region F2, 5’ end at the region F1c, 3’
end at the region B2, and the 5’ end at the region B1c. As these are the starting positions of gene replication byprimers, their end stability is important. Specifically, check to see whether the ΔG (stability) is -4.0 kcal/mol or lower.
For example the end with ΔG = -6.5 kcal/mol is more stable than the end with ΔG = -4.0 kcal/mol.
In the examples, in the set with ID number 1, the stability of the 5’ end of F1c is -3.99, so this is eliminated because
the end stability is inadequate.

For the remaining sets, any set can be selected, but if possible, primer sets with thehigher end stability should be selected.

Here, the ID numbers 5, 8, 12, 30 and 40 were selected.

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There is a “Primer Information” button above each ID Number (Figure 1.11). This button should be used todesign loop primers for the primer set selected. This will be explained further in the section on loop primer design;
click on the “Primer Information” button to save the primer information. Follow the instructions in the window to
specify the file name and location, and save the “primer information file.“ (See Figure 1.12)
This is the end of regular primer design.

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

Figure 1.11 Selected Primer Set window

Figure 1.12 Primer Information Save window21

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 V3.
Enter the target sequence file, and after confirming that “Automatic Judgment“ has been selected for the parameter
set, click on the “Primer Design“ button. (Figure not shown)

Click on the“Generate”
button.

“AT rich” hasbeen selected forthe “Parameter
Set”.

The setting includes
a longer primer
length and 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

Tm (see Figure 2.1).
Next, click on the “Generate” button to design the primers.

This will result in the generation of 1,000 primer

candidates. (Figure not shown.)
Next, click on the “Display” button to display the results of the primer design.

170 sets of primers have beenstdesigned from the 5’ end to the 3’ end, and additional sets from the 171 set have been designed again from the 5’

end to the 3’ end. (See Figure 2.2)
The method described in Section 1 is then followed (See p.13 - 18) 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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170 sets of primers have been designed from the 5’ end tost
the 3’ end and additional sets from the 171 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 automaticallyselected and the primers are designed to cover the entire target sequence.

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3. Changing the primer design conditions (parameter) (Precautions in primer
design)1.1 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 V4, 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.

Forthis reason the Tm based on the default primer length may be lower than the lower limit of default Tm value, andprevent primers from being designed.

Thus, the primer length should be increased and/ or the Tm should bedecreased.
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 acase-by-case basis, the length of each primer should be changed by one base at a time and the Tm should bechanged 1°C at a time. Once a large number of primers have been generated, then stop the adjustment andselect 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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Specify the filename and location
to save the primer
design conditions.

Figure 3.2 Saving the primer design conditions26

3.4 Using the saved primer design conditions for the primer designing
Upload the target sequence in the startup window of the PrimerExplorer V4. Next, check on “User Assignment” inthe parameter set and click on the “Browse” button to select the parameter file containing the primer design
conditions.
Click on the “Primer Design“ button.

3) Select the parameter
file containing the saved
primer design conditions.

4) Click on the “Primer
Design” button.

1) Select the target
sequence file.

2) Check on the“User Assignment”,
and click on the“Browse” button.

Figure 3.3 PrimerExplorer Ver. 4 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.
described in Section 1 (see p.13 – 18).

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The primers are selected using the procedures

“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 parameters28

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 thisbutton to specify
the location F2

Figure 4.1 Primer design window

Display of thespecified primer
location

2) Click on the
button tore-specify thelocation F2.

1) To change thespecified region, specify
a new location F2.

Figure 4.2 Window after specifying the primer location29

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 isdeleted and the newly
specified location isdisplayed.

Click on this button
to delete theinformation on thisprimer 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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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 window31

5. Loop primer design
5.1 Uploading the primer information file
Return to the PrimerExplorer V3 startup window and re-load the previously saved “primer information file.“ Clickon the “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) Click on the “Primer
Design“ button.

Figure 5.1 PrimerExplorer V3 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 window32

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“Confirm” button.

Saved primer
information
locations

1) Checkthe boxes.

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.
Examine the detailed information to choose from these loop primer sets.

Check the boxes to the left of all primer

sets, and click on the “Confirm” button to display the Primer Set Details window.

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5.2 Narrowing down the loop primer set candidates
Primer Set Details window (Figure 5.4) shows the detailed information on the six loop primer sets previously
selected.

This is the final screen for loop primer design.

ID number 1 isselectedSamesequencesSamesequences

Figure 5.4 Primer Set Details window

A close look at the forward-side loop primers will show that the ID numbers 1, 2, and 3 are the same and the ID
numbers 4, 5 and 6 are the same. As an example from the former (1 to 3), the stability of the 3’ end of the loop
primer ID number 1 is -4.74. As an example from the latter (4 to 6), the stability of the 3’ end of the loop primer ID
number 4 is -4.41.

Thus, the stability of the former three is better.

Therefore, the choices will be further narrowed

down from the former three.
Among the primers of the ID numbers 1, 2 and 3, we will now examine the stability of the 3’ end of the backward
side.

Stability is -4.46 for ID number 1, -4.06 for ID number 2, and –4.07 for ID number 3, so that the loop primer set

ID number 1 is the most stable. Among the six loop primer sets, ID number 1 is selected.

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