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Tony Rockweiler,Diagnostic Research Scientist, LucigenMarch, 2018

www.lucigen.com

Loop-Mediated Isothermal Amplification (LAMP)Primer Design and Assay Optimization

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Agenda

• LAMP Overview• Review the mechanics of LAMP• Examine the primers required and their design• Learn how to adjust software settings for better primer design• Demonstrate the effects of optimizing primer design on LAMP assay

results• Explore additional ways to optimize LAMP assays

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Introduction to LAMP

1. Amplification takes place at a single temperature (No need of a thermal cycler)

2. Uses a polymerase with high strand displacement activity (instead of Taq Polymerase)

3. Amplification is rapid (factorial as opposed to exponential in PCR)

4. Can be used for RNA templates by addition of reverse transcriptase (RT) or by using an enzyme with both RT and DNA polymerase activities

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LAMP vs. PCR

PCR LAMP

1. Requires temperature cycling Isothermal – single temperature

2. Requires 2 primers Requires 6 primers

3. Slow: Typically >1hr Rapid: Typically <30 min

4. Typical yield ~ 0.2 mg Typical yield ~ 10–20 mg

5. Not amenable to visual detection Amenable to visual detection basedon turbidity etc.

6. Sensitive to sample matrix inhibitors Tolerant to sample matrix inhibitors

7. Can be multiplexed Difficult to multiplex

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LAMP vs. PCRLAMP has Some Advantages Over PCR

• Advantages• Rapid• Sensitive and Specific • Isothermal• Less Instrument Constraints

• Disadvantages• More Difficult Primer Design• Most Detection Methods are not Sequence- specific• Difficult to Run Multi-plex LAMP• LAMP Products are not Suitable for Down Stream

Applications (i.e. cloning, sequencing)

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ApplicationsLAMP is Ideal for Certain Applications

• LAMP is Applicable for:• Detection• Point-of-Care or Field Based Detection• Limited Resource Settings• Small Test Menu • Rapid Testing

• LAMP is Inappropriate for:• Large Test Menu• Detection of Un-sequenced Targets

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Use in DiagnosticsSpeed and Simplified Instruments Make LAMP Well-suited to MDx Applications

Applications:• “Point of Care” or “Point of Collection” (POC) testing• Molecular diagnostics: e.g. infectious diseases, human or veterinary• Food testing• Environmental testing (e.g. Zika in mosquitos)• Research applications (infectious disease research)

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LAMP WorkflowEasy with Low Complexity Instrumentation

Sample

Heat lysis/NA extraction

Amplification

Detection

5-15 min

≤30 min

Real time or end point

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Detection of RNA (or DNA) Amplified by LAMPFluorescent Detection Methods are More Sensitive

• Time to Threshold (TTT) or Time to Result (TTR) provides a quantitative measure of assay performance

• Fluorescent detection can be performed as:o End-point assay/measuremento Real-time measurement (shown on left)

Signal Threshold

Tim

e to

Res

ult (

TTR

)

TTRs (time where arrows cross threshold)Reaction Time

Fluo

resc

ent S

igna

l

Fluorescent Detection (dsDNA binding Dye)

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LAMP PrimersInclusion of Loop Primers Improves LAMP Results

Figure: http://loopamp.eiken.co.jp/e/lamp/principle.html

FIP (Forward Inner Primer)F3 (Forward Outer Primer)FL (Forward Loop Primer)

BIP (Backward Inner Primer)B3 (Backward Outer Primer)BL (Backward Loop Primer)

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Amplification Intermediates

Figure: http://loopamp.eiken.co.jp/e/lamp/principle.html

Key Stem Loop DNA Structure

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Amplification Products

Cycling Amplification Steps

Figure: http://loopamp.eiken.co.jp/e/lamp/principle.html

Generation of Multimeric DNAs with Inverted Repeats During Cycling Amplification

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Primer Design OverviewKey Factors Include Tm, Length and Distances

Primer(s) Length (mer) Tm (°C)F3/B3 15-25 55-63

F2/B2 15-25 55-63

F1c/B1c 15-25 60-68

FL/BL 15-22 64-66

Distances(F2/B2) 120-160nt

Loop (F1C-F2) 40-60nt

F2-F3 0-60nt

F1c-B1C 0-100nt

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Basic Primer Design GuidelinesMultiple Characteristics Influence Primer Performance

• Primers are specified 5′ to 3′, left to right. • 40-60% GC Content• Amplicon ≤280 base pairs• Avoid runs of 3 or more of one base, or dinucleotide repeats (e.g. ACCC

or ATATATAT), both can cause mis-priming. Runs of 3 or more Gs (AGGG) may cause issues with synthesis and HPLC purification.

• Primer pairs should have similar Tms with a maximum difference of 5°C and should not be complementary to each other.

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Basic Primer Design Guidelines…continued

• Avoid regions of secondary structure; namely intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences).

• In general, select the largest ΔG value for dimer analysis minimum of −3.5 for optimal design. Select the smaller ΔG value for the ends of primers maximum of -4 for optimal design. (ΔG limits are suggestion for PrimerExplorer)

• For hairpins, the melting temperature (Tm) should be lower than the annealing temperature for the reaction; on average it should range between 55°C and 65°C. The Tm for the strongest hairpin should be at the very least 50°C and below the annealing temperature.

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Additional Tips

• Poly T linkers- e.g. GGCACATGGTCCCGTTCCTGATTTTTTAGCGCCAGACGGGATTCG

• Some reports suggest the addition of a Poly T linker in the FIP and BIP between F2-F1C and B2-B1C can improve loop formation and reaction speed.

• Mutations• Preferred not to have a mutation within a primer• Primer location least impacted by having a mutation

• 5’ ends of F3, B3, F2 and B2• 3’ ends of F1C and B1C• Internal regions

• Degenerate Nucleotides• A degenerate primer is a mix of oligonucleotide sequences in which some

positions contain a number of possible bases.

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Choosing Your Target SequenceProper Selection Ensures the Reaction will Detect Exactly What is Desired

• Strain to Strain Variation• Bacteria and viruses often have high sequence variation from strain

to strain. To detect all strains of interest, select a footprint that is conserved across strains (variants) to avoid false negatives.

• Closely Related Organisms• Closely related organisms likely to be found in the sample need to be

evaluated and care taken not to choose a conserved gene that is very similar to avoid false positives.

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To Detect Multiple Related Strains, Homologous Regions Across Strains Should be Chosen as Primer Design Targets

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Primers Designed Across Heterogenous Region Will Decrease Sensitivity for Detecting Related Strains

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Multiple Primers Sets or Degenerate Primers can be Used to Achieve Desired Multi-strain Detection

IUPAC Code R R R Y

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Avoiding Closely Related OrganismsUse BLAST to Check for Specificity

• BLAST• Blast analysis will show if the sequence you have picked is highly conserved

across other organisms. BLAST however is limited in it will only analyze species where sequence data has been deposited and should not be relied upon for specificity.

• BLAST results Summary, Top 100 Hits

LAMP Target % Coverage Total Score Expect Value

Database Target Non-target Target Non-

target Target Non-target

DENV 1 68 26-29 98-120 38-34 6.00E-05 20-320 DENV 2 66 None 67.5 None 0.004 None DENV 3 64 None 69.3 None 7.00E-04 None DENV 4 64 None 67.4 None 0.01 None ZIKAV 64 38-33 88.3 32.8-38.9 5.00E-04 11-702 CHIKV 66 None 69.3-74.7 None 5.00E-04 None

Results: None of the non-specific targets has enough identity to cross react with the LAMP primer designs

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Eiken - PrimerExplorer v5Most Widely Used Software to Design LAMP Primers

• Available for free via a web based portal on Eiken’s website(http://primerexplorer.jp/lampv5e/index.html )

• Limitations• Doesn’t design loop primers concurrently; requires a second serial

primer design execution. • Occasionally doesn’t allow for the design of one or both loop

primers• Allows for only up to 2000 bp sequences• Doesn’t support IUPAX characters other than ATCG, therefore

doesn’t handle multi-sequence alignments representations• Limited to a single execution process• Outputs only HTML

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Eiken - PrimerExplorer v5Uploading Your Target Sequence

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Eiken - PrimerExplorer v5Primer Design Settings

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Eiken - PrimerExplorer v5Avoiding Mutations/Mismatches in Your Primer Design

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Eiken - PrimerExplorer v5Common vs Specific Primers

• Common: When avoiding mutations is not possible, primers are designed allowing the inclusion of mutations. The inclusion at the following locations will allow for the detection of both mutant and wild type.

• 5’ ends of F3, B3, F2 and B2• 3’ ends of F1C and B1C• Internal regions

• Specific: When mutant and wild type need to be distinguished, primers are designed with the mutation in the locations below.

• 5’ end of F1c or B1c• 3’ end of F2 or B2• 3’ end of F3 or B3

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Eiken - PrimerExplorer v5Common vs Specific Primers

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Eiken - PrimerExplorer v5Software Adjusts Primer Location Based on Design Settings

Default designs

Common designs

Specific designs

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Eiken - PrimerExplorer v5Select the Largest ΔG Value for Dimerization

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Eiken - PrimerExplorer v5ΔG for the Ends of the Primers Less than -4 Should be Discarded

Check the stability of the following regions to confirm that the ΔG is < -4.0 kcal/mol:the 3′ end at the region F2the 5′ end at the region F1cthe 3′ end at the region B2the 5′ end at the region B1c

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Eiken - PrimerExplorer v5Designing Loop Primers

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Eiken - PrimerExplorer v5Designing Loop Primers

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Eiken - PrimerExplorer v5Select the largest ΔG value for dimerization

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Eiken - PrimerExplorer v5Select Loop Primers with the Highest 3′ End Stability

WINNER!

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What if I don’t get any designs or too many?Change Some of the Design Parameters

1. Change the distance between F3 and B3, and/or F1c and F2

2. Change the GC% setting

3. Change the target range by selecting on the sequence box and click on "within F2-B2" or "between F1c-B1c" (if you are setting the range)

4. Select a different target site

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Eiken - PrimerExplorer v5Selecting ‘Detailed Setting’ Provides Design Parameter Flexibility

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Premier BiosoftAlternative Design Software Provides a Simpler Solution

• Available for purchase• Does not have the limitations of the Eiken Software• Automatically interprets BLAST search results • Free energies can be checked for multiplexing• Local database and project to project management

(http://www.premierbiosoft.com/isothermal/lamp.html )

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Lucigen LAMP Products

Product Cat No.LavaLAMP™ DNA Master Mix 30066-1/30067-1LavaLAMP™ DNA Component Kit 30076-1/30077-1

LavaLAMP™ RNA Master Mix 30086-1/30087-1

LavaLAMP™ RNA Component Kit 30096-1/30097-1

OmniAmp™ RNA & DNA LAMP Kit 30065-1/2

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LavaLAMP™ DNA/RNA Master MixesSensitive & Fast RNA LAMP in a Master Mix Format

Master Mix Format: Streamlines reaction setup while reducing potential handling errors

Minimal Optimization: Focuses optimization on the two critical reaction parameters -primer design and reaction temperature

Lyophilization-ready: Avoids redesign of assays to remove components known to inhibit lyophilization – all components are lyophilization-compatible

Higher Reaction Temperature (68°C – 74°C): Improves primer specificity and reduces background amplification depending on the target.

Customizable: Once primers are optimized with the LavaLAMP™ Master Mix, we can work with you and your customers to generate bulk reagents that match their specific needs

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LavaLAMP™ DNA/RNA Component KitsFast, Sensitive, Thermostable LAMP in Fully Optimizable Kit Format68-74°C Reaction Temperature: Reduces background amplification and improves primer specificity depending on the target

Lyophilization-ready: Component formulations enable generation of room temperature stable test kits through lyophilization

Custom/Bulk Available: Custom volumes and dispensing available to match your specific requirements

Equivalent Performance to the LavaLAMP™ DNA/RNA Master Mix• When LavaLAMP™ DNA/RNA Component Kit and Master Mix reactions are

formulated identically

• Provides a reference point – same results – when switching from the LavaLAMP™ DNA Master Mix or RNA Master Mix to either of the component kits for additional optimization experiments

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LavaLAMP™ Enzymes vs. Bst-like EnzymesEnzymes have Different Temperature Optima

PrimerExplorer (http://primerexplorer.jp/lampv5e/index.html )LAMP Designer (http://www.premierbiosoft.com/isothermal/lamp.html )

Both software were designed with Bst or Bst like enzyme in mind

Bst and Gsp DNA polymerase works best around 60°C-65°C

LavaLAMP enzymes works best around 68°C-74°C

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Not All Primer Designs Work with Every Enzyme

0

10

20

30

40

50

60

70

68 68.4 69.2 70.4 71.8 73 73.7 74 68 68.4 69.2 70.4 71.8 73 73.7 74

No Template Control Positive

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e to

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ult (

min

)

Assay Temperature

Example 2: Published Primers

0

10

20

30

40

50

60

70

68.0 68.4 69.2 70.4 71.8 73.0 73.7 74.0 68.0 68.4 69.2 70.4 71.8 73.0 73.7 74.0

No Template Control Positive

Tim

e to

Res

ult (

min

)

Assay Temperature

Example 1: Published Primers

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What is the optimum LAMP primer Tm for LavaLAMP Enzyme?

Set IA, B, C

Set IID, E, F

Set IIIG, H, I

Set IVJ, K, L

Set VM, N, O

Set VIP, Q, R

Set VIIS, T, U

F1c and B1c 59 C 61 C 63 C 65 C 67 C 69 C 71 C

F3, F2, B3 and B2 54 C 56 C 58 C 60 C 62 C 64 C 66 C

FL and BL 56 C 58 C 60 C 62 C 64 C 66 C 68 C

Default settings, Normal GC Content

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Positive Reactions by Assay TemperatureIncreased Tm Setpoint has Faster and More Consistent Times to Result for Positive Samples Across Multiple Designs

Group I (Tm=59) Group IV (Tm=65) Group VII (Tm=71)

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Negative Reactions by Assay TemperatureIncreased Tm Setpoint More Consistent Times to Result for Negative Samples Across Multiple Designs

Group I (Tm=59) Group IV (Tm=65) Group VII (Tm=71)

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TTR Difference (NEG-POS) by primer TmIncreased Tm Setpoint has Improved Resolution Between Negatives and Positives

Betterseparation

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Set IA, B, C

Set IID, E, F

Set IIIG, H, I

Set IVJ, K, L

Set VM, N, O

Set VIP, Q, R

Set VIIS, T, U

F1c and B1c 59 C 61 C 63 C 65 C 67 C 69 C 71 C

F3, F2, B3 and B2 54 C 56 C 58 C 60 C 62 C 64 C 66 C

FL and BL 56 C 58 C 60 C 62 C 64 C 66 C 68 C

What is the optimum LAMP primer Tm for LavaLAMPEnzyme?Increasing Tm Setpoints by as much as 6° can Improve Primer Designs

Default settings, Normal GC Content

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Goals for OptimizationFaster Time to Result and Lower Background Amplification

• Increased reaction speed (faster TTR from POS)• Decreased non-specific amplification (slower TTR from NEG)• Increased separation between Positive and Negative TTR• Improved sensitivity (Detect low copy inputs)

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Factors for OptimizationMultiple Factors Need to be Assessed

• Primers

Design, Concentration, Ratio

• Reaction temperature

• Magnesium concentration

• Enzyme (concentration)

• Reaction pH

• Additives (e.g. Betaine or Triton)

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0

10

20

30

40

50

60

74°C

73.7

°C73

°C71

.8°C

70.4

°C69

.2°C

68.4

°C68

°C74

°C73

.7°C

73°C

71.8

°C70

.4°C

69.2

°C68

.4°C

68°C

74°C

73.7

°C73

°C71

.8°C

70.4

°C69

.2°C

68.4

°C68

°C74

°C73

.7°C

73°C

71.8

°C70

.4°C

69.2

°C68

.4°C

68°C

74°C

73.7

°C73

°C71

.8°C

70.4

°C69

.2°C

68.4

°C68

°C74

°C73

.7°C

73°C

71.8

°C70

.4°C

69.2

°C68

.4°C

68°C

NTC Pos NTC Pos NTC Pos

Set 1 Set 2 Set 3

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min

)

Primer Screen/Temp Opt

Primer Screen/Temperature OptimizationNot all Primer Designs are Guaranteed to Work

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Optimizing Selected Primer DesignDesign of Experiments may help in Optimization

Set Objective

Select Variables

Select an Experimental

Design

Execute the DesignCheck Data

Analyze and Interpret Results

Use Results

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Summary• LAMP is an isothermal nucleic acid amplification technique appropriate for

detection in point-of-care or low resource settings when rapid detection is necessary.

• LAMP primer design is more complex and less predictable than PCR. Screening of multiple LAMP primer sets is necessary to identify a successful design.

• Discriminative target selection and subsequent LAMP primer design guidelines are necessary for the creation of highly sensitive and specific LAMP assays.

• Adjusting Tm setpoints during primer design to match the DNA polymerase’s temperature optima can improve assay performance.

• Systematic optimization of the reaction conditions using Design of Experiments can expedite LAMP assay development.

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Resources Available

eSlides:

LAMP Webinar: http://www.lucigen.com/webinars.html#lamp

Publications:

LAMP and Isothermal AmplificationWebsite:

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QuestionsPlease Do Not Hesitate to Contact Tech Support

Thank You for Listening-in Today!

Product ManagerKaren Kleman, Ph.D.Product Manager, Amplificationkkleman@lucigen.com

Lucigen Tech Supporttechsupport@lucigen.com(608) 831-90118 am – 5 pm central time