A Guide to LAMP primer designing(PrimerExplorer V5)

Eiken Chemical Co., Ltd.

Contents
Key factors in designing LAMP primers
1. The LAMP primer
2. Key factors in the LAMP primer design
3. The steps in LAMP primer design
4. PrimerExplorer functions

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Explanations of the PrimerExplorer V5 window
1. Explanations of standard primer design window (Easy Mode)
2. Detailed design window (Expert Mode)
3. Explanations of loop primer design window (Easy Mode)
4. Detailed design window (Expert Mode)

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How to use the PrimerExplorer V51. Primer design using M13 as the template (Target)
1.1 Uploading the target sequence
1.2 Designing the primer (Easy Mode)
1.3 Designing the primer (Expert Mode)
1.4 Displaying the results
1.5 Primer set selection
2. Primer design for AT-rich sequences
3. Changing the primer design conditions (parameter) (Precaution in primer design
3.1 When too many primer sets are generated
3.2 When too few primer sets are generated
3.3 Changing and storing the primer design conditions
3.4 Using the saved primer design conditions to design primers
4. Designing primers with specified primer locations
4.1 Specifying the primer locations in the target sequence
4.2 Specify the primer location to be designated for primer design
5. Loop primer design
5.1 Uploading the primer data file
5.2 Designing loop primers
5.3 Detailed information on the loop primer sets

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Advanced primer design
6. Designing wild-type and mutant primers
6.1 Detecting wild type and mutant by amplification using common primers
6.2 Highly specific primers (specific primers that distinguish wild-type from mutant)7. Primer design that takes the location of mutation into account
7.1 Uploading the target sequence
7.2 Designing primers that do not include the mutation by entering the location ofthe mutation over the target sequence
7.3 Designing primers with mutation at the 5’ end or 3’ end of the primer location
8. Designing common primer using multiple alignment results
9. Specific primer design
9.1 Design in Easy Mode
9.2 Design in Expert Mode

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Experimental procedures and techniques
Precaution for the LAMP experiment
Reaction condition
Confirmation of the LAMP product 1
Confirmation of the LAMP product 2
Precaution for the reagent

List of terms

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Key factors in designing LAMP primer

21. The LAMP primer
The design of LAMP primers is based on the six
regions in the target sequence, designated in theFigure on the right from the 5’-end as F3, F2, F1,
B1, B2, and B3.
Forward Inner Primer (FIP) consists of the F2
sequence (at its 3’ end) that is complementary tothe F2c region, and the same sequence as F1cregion at its 5’ end.
Furthermore, Forward loop primer is designed
using the complementary strand corresponding tothe region between F1 and F2, while Backward
loop primer is designed using the complementarystrand corresponding to the region between B1and B2.

2. Key factors in the LAMP primer design
The four key factors in the LAMP primer design are the Tm, stability at the end of each primers, GC content, andsecondary structure.
2.1 Tm
Tm is estimated using the Nearest-Neighbor method. This method is currently considered to be theapproximation method that gives the value closest to the actual value.
The calculated Tm is affected by experimental conditions such as the salt concentration and oligo concentration,
so it is preferred that Tm be calculated under fixed experimental conditions (oligo concentration at 0.1 µM, sodium
ion concentration at 50 mM, magnesium ion concentration at 4 mM).
The Tm for each region is designed to be about 65°C (64 - 66°C) for F1c and B1c, about 60°C (59 - 61°C) for F2,B2, F3, and B3, and about 65°C (64 - 66°C) for the loop primers.
2.2 Stability at the end of the primers
The end of the primers serves as the starting point of the DNA synthesis and thus must have certain degree ofstability. The 3’ ends of F2/B2, F3/B3, and LF/LB and the 5’ end of F1c/B1c are designed so that the free energy
is –4 kcal/ mol or less. The 5’ end of F1c after amplification corresponds to the 3’ end of F1, so that stability isimportant. (See lower left Figure).
The change in free energy (ΔG) is the difference between the product free energy and the reactant free energy.

1The reaction proceeds toward a negative change in free energy (ΔG). The annealing between the primer and thetarget gene is an equilibrium reaction, and the annealing reaction proceeds with a smaller ΔG (see lower right
Figure on the previous page).
2.3 GC content
Primers are designed so that their GC content is between about 40% to 65%.
Primers with GC content between 50% and 60% tend to give relatively good primers.
2.4 Secondary structure
It is important, particularly for the Inner primer, that primers are designed so that they do not form secondarystructures.
To prevent the formation of primer dimers, it is also important to ensure that the 3’ ends are not complementary.
2.5 Distance between primers
The primers are designed so that the distancefrom the end of F2 to the end of B2 (the region
amplified by the LAMP method) is between 120
bases and 160 bases.
The primers are also designed so that the distancefrom the 5’ end of F2 to the 5’ end of F1 (the portion
that forms the loop) is between 40 bases and 60
bases. The primers are also designed so that thedistance between F2 and F3 is between 0 to 60
bases.

3. The steps in LAMP primer design
As indicated by the figure on the right, the steps
in primer design involve designing the regular
LAMP primers (FIP, BIP, F3, and B3) and using
them in an actual amplification. They are thenchosen as the LAMP primers if the amplification
actually proceeds and the results are satisfactory. If
the amplification does not occur or if the results
are not satisfactory, the primers need to be redesigned.
When designing the loop primers, the loopprimers are designed using the primer information
file of the selected LAMP primers. If upon
performing

theactualreaction

therateofamplification increases, then they are chosen as
the loop primers. If the results are not satisfactory, the primers need to be re-designed. The loop primers are notthe essential requirement for LAMP.

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4. PrimerExplorer functions
Currently, the two versions of Primer Explorer are available. The following table compares the functions of twoversions.
VersionFunction

Primer Explorer V4

Primer Explorer V5

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Switching between Easy and Expert Modes
Automatically narrowing down andprioritizing the primer set candidates
Standard design methods
Automatic determination of the primer
design conditions
Design that takes the location of mutation
into account
Designing primers
primer locations

with specified

Loop primer design
Primer design for the entire target region
Automatically designing common primers
Automatically designing specific primers
Inputting multiple alignment results
Saving primer set lists
Saving/uploading target sequence
information
Check of the primer ends
Saving the primer set sequence information
The individual functions are discussed below.
4.1 Easy Mode and Expert Mode

Easy Mode eliminates the need to change parameters, and displays five primer sets that are likely to havehigh amplification efficiency. It automatically narrows down and prioritizes the primer set candidates. Expert
Mode is designed for primer set customization, allowing the user to change parameters and to specify thenumber of primer sets to be designed.
4.2 Standard method
The user enters the primer design conditions to design the primers. The primer design conditions for a normal
sequence (45 %< GC<60%) has been entered as a default setting. If the target sequences are AT rich (GCcontent < 45%) or GC rich sequences (GC content > 60%), then the primers are designed with the Tm, Length,
and GC content set as follows.
Tm (ºC)

Length (mer)GC content (%)

AT rich

>55

18-25

<45GC rich

<68

15-22>603

4.3 Automatic judgment
The steps in the automatic judgment areexplained briefly in the Figure on the right.
Whenthetarget

sequenceisloaded,

The PrimerExplorer determines automaticallythe GC content of the target sequence. Based onthe result, the sequence is classified as an AT
rich sequence (GC% <45), normal sequence (45< GC %< 60), or GC rich sequence (GC %>60), and the primer design conditions areautomatically selected. The design conditions
are such that the Tm, Length, and GC content
are set to fulfill conditions that have been
optimized for a sequence, so that there is noneed for the user to enter these values.
4.4 Primer design for the entire target region
It is now possible to design primers for theentire target region. When conducting the primer
design, the primers are designed for FIP-BIPand F3 and B3 in the entire target region. Next,for each FIP- BIP region, F3 and B3 are selected
to form a primer set. The generation of primer
sets, which consist a combination of FIP-BIP with the F3 and B3, begins at the 5’ end and proceeds until the 3’end is reached. Then, the primer design proceeds again from the 5’ end to the 3’ end, and each FIP- BIP canform primer set with a maximum of three combinations of F3-B3. For each primer set with the same FIP-BIP
region, various primer sets are designed for the entire target region.
4.5 Primer design that specifies the primer location
This function permits specification of the region of each primer (F3, F2, F1, B1, B2, or B3) used in LAMP. Thisfunction is used if the region to be amplified or the regions of primers are known to be effective.
4.6 Loop primer design
After the regular LAMP primer set (FIP, BIP, F3, and B3) has been determined, the loop primers, which
reduce the amplification time and improve the specificity, can be designed. The loop primers are designed basedon the primer information file of the regular primer set.
4.7 Primer design that takes the location of mutation into accountWhen designing primers for mutations, the default option generates primers that are designed randomly, sothat the primers designed may contain the mutation itself. In general, to amplify and detect the wild type and themutation using common primers, select the primer sets whose sequence does not include the mutation point.
Under such circumstances, the primer design function that does not include mutation is used. If noappropriate primers are designed when this function is used, then the primers would be designed under lessstringent conditions that allow the mutation to be included in the 5' end or the 3’ end. It is possible to specify theprimer regions allowing mutations and the position of the mutation at that region (5’ end, internal, 3’ end).

44.8 Inputting multiple alignments
The PrimerExplorer can design two kinds of primers:
one that can detect a set of multiple genes with variousmutations (common primers) and another that can amplify
only specific gene (specific primers). During the primer
design phase, the program can input the results of multiplealignments of genes as they are. With reference to genes
at the top sequence of the alignment, the program canidentify mutation sites in each sequence and designprimers as indicated at those sites.
4.9 Automatic design of common primer
By introducing mutations into the target sequence oruploading multiple alignment results, The
PrimerExplorer enables automatic design of primers
in which the mutation sites will have little effect onamplification (common primers).
4.10 Automatic design of specific primer
By introducing mutations into the target sequence oruploading multiple alignment results, ThePrimerExplorer enables automatic design of primersthat recognize mutation sites at the end of theirsequences (specific primers).

4.11 Saving the Primer Set Design Result window
A list of primer design results can be downloaded in an Excel file. The positions of the designed primers
compared with the target sequence are displayed.
4.12 Saving the target sequence information
The PrimerExplorer can save information on the introduced mutations and information on the specified fixedprimers, along with the gene sequence information. It is also possible to re-upload saved sequences to resume
designing the primers.
4.13 Saving the primer design conditions
The primer design conditions can be saved and reloaded. Previously obtained data can be quickly displayed
by inputting the old sequence information and reloading the design conditions used to obtain the information.
4.14 Check of the primer ends
The primer’s ends are checked automatically, and thoseprimer sets possessing the complementary sequences or
specialsequencescomplementaryaresequenceautomaticallyeliminated.

isasdefinedAsymmetric

sequences (for example CCCGGG and GAATTC) andspecial sequences (for example, sequences containing thesame nucleotide at the end such as CCGGGG andAATTTT).

These can form primer dimers and thus are5eliminated at the primer design step.
Complementarity against the target sequence is also checked. The ends of the primer candidates
designed are compared to the target gene sequences, and if the end sequences of the primer candidates
also exist in a location other than the amplification region of the target sequence, then that primer set
is eliminated. Thisserves to eliminate primer sets that can cause nonspecific amplification.

4.15 Saving the primer set sequence information (ready in V5)
The primer sequence information can be downloaded in an Excel file. Basic information such as
sequences and Tm values are displayed.

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Explanation of the PrimerExplorer V5 window

Explanation of standard primer design window

Basic Design Window

Target Sequence Viewing Area：
Display the target sequence and specifies
mutation sites and primer locations.

Set Mutation：
Specifies mutationsites.

Fixed Primer：
Specifies primer locations.

The Message Viewing Area：
Display a message from the LAMP primer design
support software.

Generate sets：
Number of the primer
sets generated.

Generate：Generates primer sets.

Design Option：
Specifies common/specific primer
conditions.
Save Target：
Saves information in the Target
Sequence Viewing Area.

Display：Displays a list of the primers.

Detail Settings：
Jumps to the Detailed Design7

Detailed Design Window
Set Mutation：
Specifies mutation sites.

Target Sequence Viewing Area：
Display the target sequence and specifies mutationsites and primer locations.

Fixed Primer：
Specifies primer locations.
Save Target：
Saves information in theTarget Sequence Viewing Area.
Design Option：
Specifies common/specificprimer conditions.

Select Range ：Specifies an amplification
range.

The Message Viewing Area：
Display a message from the LAMP primer design
support software.
Sorting Rule：
Sort primer sets for output.
Default is “None”

Generate：
Generates primersets.
Display：
Displays a list of the primers.

Save Parameters：
Saves the parameter

Basic Designing：
Jumps to the Basic Design
Window.

Reset Parameters：
Resets theParameter Conditions：
Changes parameter settings.

Length：Specifies the shortest and longest lengths of each

Generate sets：
Number of the primer
sets generated.

Tm：Specifies the lowest and highest Tm of each primer.

GC rate：Specifies an acceptable range of the GC contents in each primer
Show Page ：Specifies a page to beshown.

dG threshold：Specifies a dG threshold for 5’- or 3’-end stabilityor checking dimmer formation capability.

Distance：Specifies the distance between primers

Limitations：
Specifies the number of combinations of primers togenerate a primer set and places an upper limit on
the number of sets to be generated.
Mutation/Consensus：
Handles mutation sites by setting peculiarity level(highest at the top). Capable of specifying whether toallow mutation at each of 5’, 3’ and internal sites ofeach Primer piece.

Reset Parameters：
Resets the parameters8Explanation of loop primer design window

Basic Design Window

Target Sequence Viewing Area：
Display the target sequence and specifies
mutation sites and primer locations.

The Message Viewing Area：
Display a message from the LAMP primer design support software.

Generate sets：
Number of the primer sets
generated.

Generate：Generates loop primers

Display：
Displays a list of the loop primers.

Detail Settings：
Jumps to the Detailed Design Window.

9Show Page ：Specifies a page to be shown.

Detailed Design Window

Target Sequence Viewing Area：
Display the target sequence and specifies
mutation sites and primer locations.

The Message Viewing Area：
Display a message from the LAMP primer design support software.
Generate：
Generates loop primers

Generate sets：
Number of the primer
sets generated.

Display：
Displays a list of theloop primers.

Show Page ：Specifies a page tobe shown.

Basic Designing：
Basic Designing：
Jumps to the Basic
Design Window.

Reset Parameters：
Resets the parameters

Length：Specifies the shortest and longest lengthsof each primer.
Tm：Specifies the lowest and highest Tm of each primer.

GC rate：Specifies an acceptable range of the GC contentsdG threshold：Specifies a dG threshold for 5’- or 3’-end
stability or checking dimmer formation capability.
Limitations：
Specifies the number of combinations of primers togenerate a primer set and places an upper limit on thenumber of sets to be generated.

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