LAMP primer designing software PrimerExplorer

	[1]Introduction (operating environment)
	[2]Operation of each screen (designing regular primers/designing loop primers)
	[3]Appendix

2. Operation of each screen (designing regular primers)

to designing loop primers

1. Enter the target sequence information

Upload the target sequence file for primer generation

Following formats can be used

Plain text format(sequence only)
FASTA format
GenBank format

Click on [Browse] button to choose the target sequence file on your computer.
Following options are available for the primer designing parameter setting

Automatic Judgment
Based on the GC content of the target sequence, the parameter value is automatically selected from 3 types of parameter settings. When GC content is ≤ 40% or ≥ 60%, the parameter for AT rich sequence or GC rich sequences is selected, respectively. When GC content is between 40% and 60%, the standard parameter value is chosen.
Normal
The standard parameter value is present as the default setting.
User Assignment
Click on [Browse] button and choose user's parameter file saved in user's personal computer, which is to be used as the default condition. The parameter file can be used again when it is saved as PrimerExplorer Ver.3 file. When invalid values are set as parameters, such values will not be accepted and will be ignored. And when the parameter values are not specified, "Normal" (standard parameter value) is employed.

Click on [Primer Design] button in the initial screen to proceed to the LAMP primer designing screen.

2. Primer design

Basic procedure for the primer design is as follows:

Select the [Range option].
Click on [Generate] button to generate the primer candidates.
Select the page number for the display of primer list and click on [Display] button.

Primer designing page

(1)

[Range Option]

Appoint the target sequence range (target range)

Ignore Range;

Do not specify the target sequence range

Within F2-B2;

Target region is within F2-B2 region (including F2 and B2)

Between F1c-B1c;

Target region is within F1c-B1c region but does not include F1c or B1c region

Targeting Range;

Target Range

(2)

[Generate]

Generate the primer sets

(3)

[Number of Primers]

Show the number of each primer set candidates (F1c, F2, F3, B1c, B2, B3, FIP, BIP) that meet the primer designing condition.

(4)

[F2/B2 List]

List the F2 and B2 candidates that satisfy with the primer designing condition

(5)

[Display]

List the generated primer sets in a new window

(6)

[Page]

Select the page number for the display of primer list in a new window.

(7)

[Sorting Rule]

Define the sorting pattern for the generated primer sets.

Dimer

Based on the dimer forming potential (in order of lower to higher)

F2 5'position

Listed according to the sequence position of F2 5' end position from 5' end of the target sequence

F2-B2 range

Based on the length between F2 and B2 in the order of shortest to longest

Mutation(S)
[ascendant order]

Based on the number of the primers containing
the mutation site (s) in ascending order

Mutation(S)
[descendent order]

Based on the number of the primers containing
the mutation site (s) in descending order

5'/3' edge stability

According to the end stability in the order of lower to higher

Random

In a random order

(8)

[Set Mutation]

Select the mutation sites on the target sequence, and primers are generated so as not to include the selected mutation sites. Highlight the mutation sequence and then click on [Mut/Cons] button. When the sequence is selected as mutation, the symbol "*" under the sequence is turned into "-". By clicking on [Clear] button, mutation set is cleared.

(9)

[Fixed Primer]

Fix the primer positions (s) (F1c, F2, F3, B1c, B2 or B3) and design
the primer sets with the fixed primers (s). Selected primer regions are underlined with the name of the fixed primer.

Specify the parameter setting by inputting the appropriate value or clicking on [-] or [+] button beside the number entry field to change the input value. Setting parameter condition can be saved in user's personal computer and used for primer designing later.

(10)

[Parameter Set]

Select the parameter condition

Normal

Standard parameter condition

AT rich

Parameter condition for AT rich sequence

GC rich

Parameter condition for GC rich sequence

Custom

User designed parameter
This item is available only when the user selects "User Assignment"

(11)

[Save Parameters]

The current parameter settings can be saved as files, so as to be used in
other primer designing later on.

Basic Setting

(12)

[Length]

Minimum and Maximum primer length

(13)

[Tm]

Minimum and Maximum primer Tm value

(14)

[Distances]

Minimum and Maximum length between primers (primer position)

(F2-B2)

F2-B2 (including both primer length), amplified region

Loop(F1c-F2)

F1c (B1c) -F2 (B2) (including F2 (B2)),loop length

F2-F3

F2 (B2) -F3 (B3) (exclude the primer itself)

F1c-B1c

F1c - B1c (exclude the primer itself)

Advanced Setting

(15)

[Limitations]

Maximum number of combinations when primer set is generated.

F1c/B1c

The number of F1c (B1c) candidates, which can combine with each F2 (B2) when generating FIP (BIP)

F2/B2

The number of F2 (B2) candidates, which can combine with each F1c (B1c) when generating FIP (BIP).

F3/B3

The number of F3/B3 (outer primer) candidates, which can combine with one FIP/BIP set.

Sets

Number of the finally generated primer sets.

(16)

[dG threshold[Kcal/mol]]

dG threshold
Generated primers that meet the following criteria are selected as candidates

5'stability

Its 5' stability value (dG) is below this value.

3'stability

Its 3' stability value (dG) is below this value.

dimer check

The primer's dimer forming energy (dG) (stability between
the combined primers) is more than this value.

(17)

[Mutation/Consensus]

When this item is selected, software will allow mutant to exist in 3' or 5'end. (In the default condition, primer sets are generated so as not to include any mutation sites).

(18)

[GC rate(%)]

Acceptable value for GC rate

(19)

[Experimental Conditions]

Concentration of

oligo[microM]

Oligo-nucleotide (µM)

Na⁺[mM]

Sodium ion (mM)

Mg²⁺[mM]

Magnesium ion (mM)

(20)

Reset Parameter

Reset parameter value to the default condition.

Caution
The target sequence must be less than 2,000bp in length. When more than 2,000bp of the target sequence is uploaded, the primer designing can not be performed and the following page will appear, in which the message " Target sequence length is too long! Please input the sequence less than 2,000bp" is shown. The parameter setting cannot be displayed and primer design cannot be performed.

3. Generated primer list

Up to 100 generated primer sets will be listed in each page. Each row stands for one primer set, and each primer fragment's location refers to the target sequence above directly.

Check the box (left-side each primer set) and click on [Details] button (top of the page), then the detail information of the selected primers will be displayed.

4. Detail information of the selected primer sets.

The primer's information below is shown.

The following information is shown with each primer fragment.

Name
Position of 5' end on the target sequence
Position of 3' end on the target sequence
Length
Tm value
Stability of 5' end
Stability of 3' end
GC rate
Primer sequence

After checking the check-box, click on the [Confirm] button will display the details of the selected primer sets in a new window.

5. Save selected primer sets

The primer sets' information is listed, which is selected in the Primer sets Details page.

Click on [Primer Information] to save the primer information as a file in your PC. This information can be used for designing the loop primer.

	[1]Introduction (operating environment)
	[2]Operation of each screen (designing regular primers/designing loop primers)
	[3]Appendix