Loop Mediated Isothermal Amplification

Abisha.S.JFC&RI

LAMP

LAMP" stands for Loop-mediated Isothermal Amplification.

This technology was developed by Notomi et al.

It is a very sensitive, easy and time efficient method.

The LAMP reaction proceeds at a constant temperature using a strand displacement reaction.

This may be of use in future as a low cost alternative to detect certain diseases. It may be combined with a reverse transcription step to allow the detection of RNA.

TechniqueLAMP is an isothermal nucleic acid amplification technique.

In contrast to the polymerase chain reaction (PCR) technology, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler.

It is characterized by the use of the 4 different primers specifically designed to recognize 6 distinct regions on the target gene.

The reaction process proceeds at a constant temperature using Strand displacement reaction.

Amplification and detection of gene can be completed in a single step, by incubating the mixture of sample primers,DNA polymerase with strand displacement cativity and substrate at constant temperature(about 65 C⁰ )

In the target gene, the F3c,F2c and the F1c regions at the 3’ side and the B1,B2 and B3 regions at the 5’ side are the distinct regions

There is no need for a step to denature double stranded into a single stranded form

It provides high amplification effeciency with DNA being amplified 10 -10 times in 15- 60 minutes.⁹ ⁱ⁰

Because of its high specificity, the presence of amplified product can indicate the presence of the target gene.

Types of Primers used in LAMPLAMP is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions of the target gene. The four primers used are as follows

1.Forward Inner Primer (FIP):2.Forward Outer Primer (FOP): The FOP (also called F3 Primer) 3. Backward Inner Primer (BIP):4.Backward Outer Primer (BOP):

Principle;

When the target gene(DNA template) and the reagents are incubated at a constant temperature between 60-65 C, the following reaction steps proceed.⁰

Stages in Loop-mediated Isothermal Amplification

Step 1One of the LAMP primers can anneal to the complementary sequence of double stranded target DNA, then initiates DNA synthesis using the DNA polymerase with strand displacement activity, displacing and releasing a single stranded DNA.

With the LAMP method, unlike with PCR, there is no need for heat denaturation of the double stranded DNA into a single strand. The following amplification mechanism explains from when the FIP anneals to such released single stranded template DNA.

STEP2Through the activity of DNA polymerase with strand displacement activity, a DNA strand complementary to the template DNA is synthesized, starting from the 3' end of the F2 region of the FIP.

Outer primer F3 hybridizes to the F3c region of the target DNA and extends, displacing the FIP linked complementary strand. This displaced strand forms a loop at the 5' end.

Step 3 This single stranded DNA with a loop at the 5' end serves as a template for BIP.

B2 hybridizes to B2c region of the template DNA. DNA synthesis is now initiated leading to the formation of a complementary strand and opening of the 5' end loop.

Step 4. Now, the outer primer B3 hybridizes to B3c region of the target DNA and extends, displacing the BIP linked complementary strand.

This results in the formation of a dumbbell shaped DNA.

Step 5The nucleotides are added to the 3' end of F1 by DNA polymerase, which extends and opens up the loop at the 5' end. The dumbbell shaped DNA now gets converted to a stem loop structure.

This structure serves as an initiator for LAMP cycling, which is the second stage of the LAMP reaction

Step 6 To initiate LAMP cycling, the FIP hybridizes to the loop of the stem-loop DNA structure.

Strand synthesis is initiated here.

As the FIP hybridizes to the loop, the F1 strand is displaced and forms a new loop at the 3' end.

Step 7 Now nucleotides are added to the 3' end of B1. The extension takes place displacing the FIP strand.

This displaced strand again forms a dumbbell shaped DNA. Subsequent self-primed strand displacement DNA synthesis yields one complementary structure of the original stem loop DNA and one gap repaired stem loop DNA.

Step 8

Both these products then serve as template for a BIP primed strand displacement reaction in the subsequent cycles.

Thus, a LAMP target sequence is amplified 13 fold every half cycle.

The final products obtained are a mixture of stem loop DNA with various stem lengths and various cauliflower like structures with multiple loops.

The structures are formed by annealing between alternatively inverted repeats of the target sequence in the same strand.

Loop primers

The loop primers(either loop primer B or loop primer F) containing sequences complementary to the single stranded loop region( either between the B₁ and B₂ regions or between F₁ and F₂ regions) on the 5’ end of the dumbell like structure, provide an increased number of the starting points for the LAMP method.

An example is shown in the figure where there is an amplified product containing six loops. In the original LAMP method, four of these loops would not be used, but through the use of Loop Primers, all the single stranded loops can be used as starting points for DNA synthesis

The investigation on how Loop Primers affect amplification time (original method: no Loop Primer; rapid method: with Loop Primers) shows that the time required for amplification with Loop Primers is one-third to one-half of that without Loop Primer.

With the use of Loop Primers, amplification can be achieved within 30 minutes.

Lamp DetectionIn a LAMP assay, the reaction takes place in a single tube containing buffer, target DNA, DNA polymerase and primers.

The tube is incubated at 64°C in a regular laboratory water bath or heat block that helps in maintaining a constant temperature.

The amplified product can be detected by naked eye as a white precipitate or a yellow-green color solution after addition of SYBR green to the reaction tube

Advantages

1. Amplification of DNA takes place at an isothermal condition (63 to 65°C) with greater efficiency.2. Thermal denaturation of double stranded DNA is not required.3. LAMP helps in specific amplification as it designs 4 primers to recognize 6 distinct regions on the target gene.4. LAMP is cost effective as it does not require special reagents or sophisticated equipment.5. This technology can be used for the amplification of RNA templates in presence of reverse transcriptase.6. LAMP assay takes less time for amplification and detection

DisadvantagesComplicated primer designTo sensitive and prone to contamination and small changes in conditionApplications1. LAMP is used in rapid diagnosis of viral, bacterial and parasitic diseases.2. It helps in the identification of genus and species-specific parasites

Efficient Primer Design for Loop-mediated Isothermal Amplification using LAMP Designer

LAMP Designer designs four primers along with two additional loop primers to identify six distinct regions.

The software automatically interprets BLAST results and even avoids homologous regions on the sequence while designing primers to prevent non-specific amplification.

The designed primers can also be BLAST searched to verify their specificity.

LAMP vs PCR

LAMP vs PCR

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