A Guide to LAMP primer designing(PrimerExplorer V4)

Eiken Chemical Co., Ltd.

_Contents
Key factors in designing LAMP primers
1. The LAMP primer

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2. 2 Key factors in the LAMP primer design1

3. The steps in LAMP primer design2

4. PrimerExplorer functions

3

Explanations of the PrimerExplorer Ver.4 window
1. Explanations of standard primer design window (Easy Mode)7

2. Detailed design window (Expert Mode)8

3. Explanations of loop primer design window (Easy Mode)9

4. Detailed design window (Expert Mode)10

How to use the PrimerExplorer V.4
1. Primer design using M13 as the template (Target)

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1.1 Uploading the target sequence

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1.2 Designing the primer (Easy Mode)12

1.3 Designing the primer (Expert Mode)15

1.4 Displaying the results

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1.5 Primer set selection20

2. Primer design for AT-rich sequences

22

3. Changing the primer design conditions (parameter) (Precaution in primer design)24

3.1 When too many primer sets are generated

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3.2 When too few primer sets are generated

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3.3 Changing and storing the primer design conditions25

3.4 Using the saved primer design conditions to design primers

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4. Designing primers with specified primer locations29

4.1 Specifying the primer locations in the target sequence29

4.2 Specify the primer location to be designated for primer design30

5. Loop primer design

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5.1 Uploading the primer data file32

5.2 Designing loop primers

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5.3 Narrowing down the loop primer set candidates

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Advanced primer design
6. Designing wild-type and mutant primers

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6.1 Detecting wild type and mutant by amplification using common primersi35

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

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39
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7.2 Designing primers that do not include the mutation by entering the location ofthe mutation over the target sequence39

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

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8.1 Reading multiple alignment results48

8.2 Designing common primers

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9. Specific primer design

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9.1 Design in Easy Mode51

9.2 Design in Expert Mode53Experimental procedures and techniques
Precaution for the LAMP experiment

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Reaction condition

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Confirmation of the LAMP product 156

Confirmation of the LAMP product 256

Precaution for the reagent

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List of terms

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Key factors in designing LAMP primer

_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 60bases.

The primers are also designed so that thedistance between F2 and F3 is between 0 to 60bases.

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 bere-designed.
When designing the loop primers, the loopprimers are designed using the primer information
file of the selected LAMP primers.

If uponperforming

rate

the actual

reaction theof

amplification 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.
versionPrimer Explorer Ver.3

Primer Explorer Ver.4

×○×○○○

○

○

○

○*○

○

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○○

Function
Switching between Easy and Expert Modes
Automatically narrowing down and prioritizing
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 with specified primer
locations* To specify primer regions, including for mutations and the locations of themutations at those regions (5’ end, internal, 3’ end).
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.

AT richTm (ºC)

Length (mer)GC content (%)

>55

18-25

<453

GC rich<6815-22>60

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

sequenceisloaded,PrimerExplorer determines automatically the GC
content of the target sequence.

Based on theresult, the sequence is classified as an AT richsequence (GC% <45), normal sequence (45 <GC %< 60), or GC rich sequence (GC %> 60),
andtheprimer

designautomatically selected.

conditions

areThe 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-BIP
and 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 Application of multiple alignment results (ready inVer.4)
PrimerExplorer Ver. 4 can design two kinds of primers:
one that can detect a set of multiple genes with various
mutations (common primers) and another that can amplify
only specific gene (specific primers). During the primer
design phase, the program can input the results of multiplealignmnts 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 (ready in Ver. 4)
By introducing mutations into the target sequence oruploading multiple alignment results, Ver. 4 enables
automatic design of primers in which the mutation sites will
have little effect on amplification (common primers).
4.10 Automatic design of specific primer (ready in Ver. 4)
By introducing mutations into the target sequence or
uploading multiple alignment results, Ver. 4 enables
automatic design of primers that recognize mutation sites
at the end of their sequences (specific primers).
4.11 Saving the primer set list (ready in Ver. 4)
Ver. 4 displays the locations of the primers designed with reference to the target sequence in a view window,
and allows for downloading the primer design results into an Excel file.
4.12 Saving the target sequence information (ready in Ver. 4)
Ver. 4 can save not only gene sequence information but also the information of introduced mutations andspecified fixed primers. It can also re-upload saved sequences to resume designing the primers.
4.13 Saving the primer design conditions
The primer design conditions that the users manually have input can be saved as a file style and re-loaded.
When the sequence and the preserved primer design condition are input, the data previously obtained can bequickly displayed and the primer design working can be resumed easily.
5.5 Check of the primer ends
The primer’s ends are checked automatically, and thoseprimer sets possessing the complementary sequences or
special sequences are automatically eliminated.
complementary sequence isA

defined as symmetricsequences (for example CCCGGG and GAATTC) andspecial sequences (for example, sequences containing
the same 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 alocation other than the amplification region of the target sequence, then that primer set is eliminated.
serves to eliminate primer sets that can cause nonspecific amplification.

6This

Explanation of the PrimerExplorer V4
window

_Explanation of standard primer design window

Basic Desigin Window

Set Mutation：
Specifies mutationsites.
Fixed Primer：
Specifies primer
locations.

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

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

Generate sets：
Number of the primer
sets generated.

Generate：Generates primer sets.

Display：Displays a list of the primers.

Detail Settings：
Jumps to the Detailed Design Window： Existing functions： New functions

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Design Option：
Specifies common/specific primer
conditions.

Save Target：
Saves information in the Target
Sequence Viewing Area.

Detailed Design Window

Set Mutation：
Specifies mutation sites.

Fixed Primer：
Specifies primer locations.
Target Sequence Viewing Area：
Displays the target sequence and specifies mutationsites and primer locations.

Select Range：
Specifies anamplification range.

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

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

Generate：
Generates primer sets.
Display：
Display a list of theprimers.

Sorting Rule：
Sort primer sets for output.
The “Easy”(default) option has
been added in the Easy Mode.

Save Parameters ：Saves the parameter set.

Basic Designing：
Jumps to the Basic
Design Window.

Reset Parameters：
Resets the parameters
Length： Specifies the shortest and longest lengths ofeach primer.

Parameter
Conditions：
Changes parametersettings.

Tm：Specifies the lowest and highest Tm of each primer.
GC rate：Specifies an acceptable range of the GC
contents in each primer

Generate sets：
Number of the primer
sets generateddG threshold：Specifies a dG threshold for 5’- or 3’-end stability orchecking dimmer formation capability.

Show Page ：Specifies a page to beshown.

Distance：Specifies the distance between primers
Limitations：
Specifies the number of combinations of primers togenerate a primer set and places an upper limit on thenumber of sets to be generated.

Mutation/Consensus：
Handles mutation sites by setting peculiarity
level (highest at the top). Capable of specifying
whether to allow mutation at each of 5’, 3’ andinternal sites of each Primer piece.

Reset Parameters：
Resets the parameters.

8Explanation of loop primer design window

Basic Design Window

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

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

Generate sets：
Number of the primer sets generated.

Generate:：Generates loop primers.

Show Page：
Specifies a page to be shown.
Display：
Displays a list of the loop primers.

Detail Settings:：
Jumps to the Detailed Design Window.

： Existing functions： New functions

9Detailed Design Window

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

Generate：Generates the loopprimers.
Display：
Displays a list of theloop primers.

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

Generate sets：
Number of the primer
sets generated.

Show Page ：Specifies a page to be shown.

Reset Parameters：
Resets the parameters.

Basic Designing：
Jumps to the Basic
Design Window.
Length ： Specifies the shortest and longest
length of each primer.
Tm：Specifies the lowest and highest Tm of each primer.
GC rate：Specifies an acceptable range of the GC
contents in each primer.
dG threshold：Specifies a dG threshold for 5’- or 3’-end stabilityor checking dimmer formation capability.
Limitations：
Specifies the number of combinations of primers
to generate a primer set and places an upper limiton the number of sets to be generated.
Distance：Specifies the distances between primers.

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