Journal club 11 06-12

INSTITUT FÜR VIROLOGIE

Loop-mediated isothermal

amplification (LAMP) of DNA

Rokshana Parvin


Nucleic acid amplification is one of the most valuable tools in

virtually all life science fields

PCR; polymerase chain reaction

NASBA; nucleic acid sequence-based amplification

SMART; signal mediated amplification of RNA technology

SDA; strand displacement amplification

RCA; rolling circle amplification

LAMP; loop-mediated isothermal amplification of DNA

HDA; helicase-dependent amplification

SPIA; single primer isothermal amplification




http://loopamp.eiken.co.jp/e/lamp/index.html



Loop mediated isothermal amplification (LAMP) is a

single tube technique for the amplification of DNA offering

rapid, accurate, and cost-effective diagnosis of infectious

diseases


─ no need for a step to denature double stranded into a single stranded form.

─ The whole amplification reaction takes place continuously under isothermal

conditions (water bath).

─ The amplification efficiency is extremely high. the amount of DNA ( 109-1010

times in 15-60 minutes) produced in LAMP is considerably higher than PCR

based amplification

─ The target sequence is amplified at a constant temperature of 60 - 65 C

using 4 primers to recognize 6 distinct regions, and a polymerase with

high strand displacement & replication activity

─ The total cost can be reduced, as it dose not require special reagents or

sophisticated equipment's (Thermal cycler).

─ Amplification also can be done with RNA templates following the same

procedure, simply through the addition of reverse transcriptase.

Characteristics


Design of primers

Design 4 types of primers based on the following 6 distinct regions of the target

gene:

F3c F2c F1c

3' 5'

At the 5' side: B1, B2 and B3 regions

B1 B2 B3

5'3'

F3 F2 F1

3'5'

B1c B2c B3c

5' 3'

Target DNA

Target DNA

At the 3' side: F3c, F2c and F1c regions


FIP Forward Inner Primer (FIP) consists of the F2

region (at the 3' end) that is complementary to

the F2c region, and the same sequence as the

F1c region at the 5' end.

F3

Primer

Forward Outer Primer consists of the F3 region

that is complementary to the F3c region.

BIP Backward Inner Primer (BIP) consists of the B2

region (at the 3' end) that is complementary to

the B2c region, and the same sequence as the

B1c region at the 5' end.

B3

Primer

Backward Outer Primer consists of the B3

region that is complementary to the B3c region.

http://loopamp.eiken.co.jp/e/lamp/primer.html

A

T

C

G

AG

GT

A

C

T

C


Reaction mixture for LAMP

0.8 µM each BIP&FIP

0.2 µM each F3&B3

400 µM dNTPs

1M betaine

20 mM Tris-HCL

10 mM KCL

10 mM (NH4) 2 SO4

4 mM MgSO4

0.1% Triton X-100

Bst DNA polymerase

Target DNA

25 µlReaction

Mixture60-65 C for 1 h

Terminate by 80-

90 C for 5-10 m


Betaine

N,N,N-trimethylglycine

Reduce base stacking,

Stimulated the overwall rate of the reaction

Increased target selectivity with a significant

reduction of irrelevent sequences

Bst DNA polymerase

Bst polymerase is a thermostable DNA Pol I that was

isolated in 1968 (Stenesh 1968) from the thermophilic

bacterium Bacillus stearothermophilus (Bst), which

proliferates between 39 and 70 C. Bst Pol I is active at

an optimal temperature of 65 C, and is inactivated after

15 min incubation at 75 C.

http://upload.wikimedia.org/wikipedia/commons/2/21/Trimethylglycine.png


1. Initial step: starting material producing step

Mechanism or Basic principle

Double stranded DNA is in the condition of dynamic

equilibrium at the temperature around 65 C, One of

the LAMP primers can anneal to the complimentary

sequence of double stranded target DNA, then

initiates DNA synthesis using the Bst DNA

polymerase with strand displacement

activity, displacing and releasing a single stranded

DNA.

STEP2

Through the activity of DNA polymerase with strand

displacement activity,initates complementary strand

synthesis

STEP1

Inner primer FIP anneal to F2c in the target DNA


STEP3

The F3 Primer anneals to the F3c region, outside of

FIP, on the target DNA and initiates strand

displacement DNA synthesis, releasing the FIP-

linked complementary strand.

STEP4

A double strand is formed from the DNA strand

synthesized from the F3 Primer and the template

DNA strand.

STEP5

The FIP-linked complementary strand is released

as a single strand which forms a stem-loop

structure at the 5' end because of the

complementary F1c and F1 regions


STEP7

Double stranded DNA is produced through the

processes described in Step (6).

STEP8

The BIP-linked complementary strand displaced

in Step (6) forms a structure with stem-loops at

each end, which looks like a dumbbell structure.

This structure serves as the starting structure for

the amplification cycle in the LAMP method

(LAMP cycling).

STEP6

This single strand DNA in Step (5) serves as a

template for BIP-initiated DNA synthesis and

subsequent B3-primed strand displacement DNA

synthesis.


2. Cycling amplification step

FIP anneals to stem- loop

primes strand displacement

Generate one stem-loop and

additional inverted copy of

target sequence in the stem

where loop form at opposite

end via BIP

Subsequent self primed strand

displacement DNA synthesis

yields one complementary

structure of original stem-loop

Thus ,in LAMP the target

sequence is amplified 3-fold

every half cycle


3. Elongation and recycling step

The final products are mixture of stem-

loop DNAs with stem lengths and

cauliflower - like structures with

multiple copies


Mechanism or Basic principle- Video

http://loopamp.eiken.co.jp/e/lamp/anim.htmlhttp://www.youtube.com/watch?v=5Wi-kkSFy48


The Loop Primers containing sequences complementary to the

single stranded loop region (either between the B1 and B2

regions, or between the F1 and F2 regions) on the 5' end of the

dumbbell-like structure, provide an increased number of

starting points for DNA synthesis for the LAMP method.

Use of loop primer

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.


Analysis of product

1. Gel elctrophoresis: 2-3% agarose gel stained with EB / SYBR green or

Calcein

2. Naked eye: Photometry for turbidity caused by increasing quantity of

Magnesium pyrophosphate in solution with or without addition of

SYBR green or manganese loaded calcein

3. Real time: either by measuring the turbidity or the signals from DNA

produced via fluorescent dyes that intercalate or directly label the

DNA, and in turn can be correlated to the number of copies initially

present. So, LAMP can also be quantitative.



A) The positive PCR reactions show a 273 bp amplicon while

A) LAMP produces a characteristic ladder of multiple bands on an agarose gel

indicating stem-loop DNA with inverted repeats of the target sequence.

http://www.sciencedirect.com/science/article/pii/S0014489410000202


Visual detection of sample by LAMP from

positive and negative samples.

(1) Naked eye detection without SYBR green;

(2) naked eye detection with SYBR green;

(3) detection under an UV trans-illuminator

using SYBR green;

(4) detection under a hand-held black light

using SYBR green.

http://www.sciencedirect.com/science/article/pii/S0001706X09002885


Simple screening assay in the field or at the point of care by clinicians

Low cost

Amplified at a constant temperature of 60 - 65 °C

Easy visualization by the naked eye

The reaction can be followed in real-time

This technology has been developed into commercially available

detection kits for a variety of pathogens including

bacteria, parasite, fungus and viruses

Uses and benefits





Thank you so much for

your kind attention


In the DNA amplification process by DNA polymerase, pyrophosphate ions are produced as a

by-product from the reaction substrate deoxyribonucleotide triphosphates (dNTPs). The calcein

in the reaction mixture initially combines with manganous ion (Mn2+) so as to remain quenched.

When the amplification reaction proceeds, manganous ion is deprived of calcein by the

generated pyrophosphate ion (P2O74- ), which results in the emission of fluorescence. And the

free calcein is apt to combine with magnesium ion (Mg2+) in the reaction mixture, so that it

strengthens the fluorescence emission.

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Journal club 11 06-12

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