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Loop-Mediated Isothermal Amplification Method (LAMP): Low and Effective cost Novel Tool for Molecular Public Health

December 9, 2009 - 05:35 -- Usa Lek-Uthai

This technique need not to have even high cost equipments (especially PCR or GelDoc or refrigerator). The specific LAMP primer set consist of F3(forward outer primer), B3 (backward outer primer), FIP (forward inner primer), and BIP (backward inner primer) These primers will be designed using the Primer Explorer program (http://www.loopamp.eiken.co.jp) to amplify the 18S ribosomal
RNA gene (http://www.ebi.ac.uk.com). The protocol could be optimized for each reaction; my experience for the study of Acanthamoeba spp rapid identification, LAMP was performed for 60 min at temp 65 C in 25 microL of a mixture containing 2 microL of extracted DNA, 40 pmol each of FIP and BIP, 5 pmol each of F3 and B3, 1.4 mM deoxynucleoside triphosphates, 0.8 M betaine and 1 microL of Bst DNA polymerase in buffer (20 mM Tris–HCl pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, and 0.1% Tween 20). The reaction was terminated by heating (using water bath or heating block) for 2 min at temp 80 C. Turbidity of the LAMP reaction will be inspected visually in real time. The DNA products could also be visualized under UV light after addition of Loopamp fluorescent detection reagent (Eiken Chemical Co., Ltd.). LAMP products was confirmed by electrophoresis in 1.5% agarose gel and visualization by staining assay. The sensitivity and specificity were 100%. (see more at: Usa Lek-Uthai et al. Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method. Experimental Parasitology Volume 121, Issue 4, April 2009, Pages 342-345). However, the study of genotyping and copy numbers for the Plasmodium spp specific genes (the species identification need not to have any DNA extraction method from fresh or dried blood spots) in different malaria endemic regions in Thailand since 2007 have been doing well (unpublished). Therefore, this technique might be useful for molecular public health in the developing country’s disease control program. LAMP took less time than qPCR, which required 2 h as compared to 1 h for the LAMP procedure. However, the cost of this technique is the cheapest (about 70 cents US per sample compared to 5–7 $US per sample using DNA amplification by realtime PCR.

Comments

Amy Mikhail's picture
Submitted by Amy Mikhail on

Hi Usa,

I have yet to try LAMP but am curious as to it's practical feasibility in the field... from the above, I gather that you still need a heat block (admittedly that is much cheaper than a thermocycler, but still needs electricity).

Also, I'm guessing that for routine use (in a monitoring / malaria sentinel site surveillance program, for example) turbidity would be difficult, so the fluorescent detection reagent would be more appropriate. I have some questions about this:
- How much does this cost?
- Is the colour change easy to see if you have a simple (and cheap) UV torch?
- Is it multi-plexable (can one have a reaction that identifies all 5 human malaria species (Pf, Pv, Pm, Po and P. knowlesi) in the same tube, and if so what would be the detection mechanism)?

Related to the last point - in countries where malaria species ID is important, if not multi-plexable in its current format that would raise the cost to $3.5 per sample. If one is selective about real time PCR reagents, one can spend as little as $1.3 per sample to detect all 5 human malaria species (this includes reagents and plastic consumables for crude DNA extraction from filter paper blood spots and real time PCR amplification only - it does not include equipment costs, but I think your $ 0.7 / sample also does not include equipment costs?).

One last question - how sensitive is the LAMP malaria assay compared to Snounou's nested PCR (I realise that above, you are talking about a genotyping assay rather than a species ID assay, but just curious if this is known for the latter)?

Amy Mikhail
Program Manager, ACTc Afghanistan Malaria Project
London School of Hygiene & Tropical Medicine

------ Address in Afghanistan ---------------------
Health Protection & Research Organisation
House number P860
Street 10
Taimany
District

Usa Lek-Uthai's picture
Submitted by Usa Lek-Uthai on

Dear Amy
Thanks for your comment. I also need to use a heating block (in my country, Thailand , there are feasibility to use an electricity, at all village level, i meant at the whole country, so this LAMP might have experienced to the villager volunteer to train for this and possible to set this lab with a higher sensitive than microscopy. I have compared and checked myself and my colleague to identify the speciation among naked eyes (no UV lamp) and fluorescence inspection (using a simple lamp source). They presented no different of the precipitation ratio and color. Actually i am learning by doing, what ever u ‘d like to discuss pls feel free with my great thanks. The FDR (for LAMP) u can order they have ready use and powder, pls see wavelength (I meant emiss and excite) the cost is depend on the company or import order (Japan), - Is the colour change easy to see if you have a simple (and cheap) UV torch? The color is due to the originate source (brink green or orange). - Is it multi-plexable (can one have a reaction that identifies all 5 human malaria species (Pf, Pv, Pm, Po and P. knowlesi) in the same tube, and if so what would be the detection mechanism)? There have been done nested, but for multiplex PCR, i have not yet tried, because they need to design a universal primer for this LAMP (nobody do it), and the FP and BP for all 4 species (no Pk) had a report, may be u can try, the contra-indication for this LAMP especially the nested PCR product could not do in LAMP reaction.
- it does not include equipment costs, but I think your $ 0.7 / sample also does not include equipment costs?)..yes it has just tangible cost without equipment and intangible cost. One last question - how sensitive is the LAMP malaria assay compared to Snounou's nested PCR (I realise that above, you are talking about a genotyping assay rather than a species ID assay, but just curious if this is known for the latter)? I had put other study for you, In 2007, there have a report for the LAMP method to detect all 4 human Plasmodium spp (no Pk). They evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. other study from the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Japan identified Plasmodium-carrying mosquitoes, specifically a Plasmodium-transmitting experimental model using rodent malaria parasite (Plasmodium berghei) and anopheline mosquitoes (Anopheles stephensi). The detection sensitivity limit of the LAMP reaction amplifying the SPECT2 gene was determined to be 1 x 10(2) purified Plasmodium parasites, estimated to be sufficient for reliable identification of infectious mosquitoes. The robustness of the LAMP reaction was revealed by its ability to detect both Plasmodium oocysts and sporozoites from an "all-in-one" template using whole mosquito bodies. Moreover, LAMP successfully identified an infectious mosquito carrying just a single oocyst in its midgut, a level that can be easily overlooked in conventional microscopic analysis. These observations suggest that LAMP is more reliable and useful for routine diagnosis of vector mosquitoes in regions where vector-borne diseases such as malaria are endemic.
By the way, for myself I had compared this LAMP without turbidity meter (for public health field) to Realtime PCR (SYBR) using Pf-B -actin1 (a copy no) to be a control gene anyhow we are analysing the percent copy no, due to the calculated formular. This LAMP had first reported by the Japanese:)) and i had learnt from Prof. Suzuki (Hokkaido U), You could see more informations from the web.
Cheers
Usa

Usa Lek-Uthai,
Mahidol University,
Bangkok, Thailand

Submitted by Guest (not verified) on

Dear Usa,

I am working at Molecular Biology Lab. our project contain LAMP method. We have done well LAMP reaction with DNA genomic tamplate, now we want to use Palsmid which include target gene (we inserted target gene by cloning and check by digesting with retriction enzyme, result is good band) is possitive control but we have problem. That is when we run LAMP reactions with genomic DNA and plasmid DNA with the same marterials, result Lamp with genomic DNA is ok, esle not ok. I do not understand why.
- next question: we did not see pticipitation after endding reaction but if we added more MgSO4 1M and EtBr 10% to product tube then see under UV light, we can see possitive product is bright and nagative is dark. would you tell me that I did right?can we detect LAMP product by this way?

Submitted by shesheer (not verified) on

I too faced similar problem when I used cloned gene of MTB in a plasmid for sensitivity assays. Only the stock plasmid of miniprep is being picked up but not the subsequent dilutions. But it`s working well with the clinical samples and to a detection limit lower than nested PCR.

Does anybody has any suggestion

Usa Lek-Uthai's picture
Submitted by Usa Lek-Uthai on

As the SYBR green I dye add to tube after the LAMP reaction, the color will change to yellowish green in a positive reaction and remain reddish orange in the negative reaction. The problem of color changed when negative control is visualed from orange to green. I thought that it is possible to have a high risk of amplicon contamination since the tube have to be opened to add the dye?. so to reduce the chances of contamination, similar protocols to those followed for PCR will be required. The color change may occur earlier at higher viral loads such as 10E7 copies and may take longer for lower loads (60 min for 10,000 to 100,000 copies/ml). So, the negative control shrimp may be contaminated. The LAMP test should theoretically not amplify nontarget sequences, so design a set of primers would be specified carefully with no cross reaction to other contaminated agents, (BLAST etc)). In this case I suggest that you have to do confirmation by PCR using the same primers set.

Usa Lek-Uthai,
Mahidol University,
Bangkok, Thailand

Submitted by Guest (not verified) on

Dear Usa,
I am studying at Mahidol University. My thesis is about Loop mediated isothermal. I have a problem about this. I brought Lamp product to comfirmed by electrophoresis.I don't understand what did it mean? The picture show that the band didn't ladder. I think my products were not contaminate.Because negative control is empty(I cant see anything in this). I would like to send the picture to you but i can't. Would you mind I ask you about your Email or you can contact me at beau_pt@hotmail.com.
Finally, I am looking forward for your answer to me. Thank you veru much
Sincerely yours,
Kanokporn

Submitted by Guest (not verified) on

Yes, the negative lane should n't see any, by the way i have sent you an email address last month, you can send me your image and tell me your optimization process (?temp) or any help? and please confirm the 4 primers design. Shortly, LAMP amplified product is different from others PCR products, The amplified products have various size structures consisting of alternately inverted repeats of the target sequence on the same strand, remember no denature for this method!. the temperature have to stable all the time reaction!
cheers
Usa

Submitted by Guest (not verified) on

Dear Usa

I am trying to standadise LAMP in my lab but i am getting certain problem in negative control.There is a good ladder pattern of bands in positive control but the negative control is also showing ladder pattern of bands provided the pattern of bands is different in positive and negative control.To rule out contamination i have changed almost all the reagents and performed the reaction in laminar flow everytime but still band pattern in negative control persisted.Distilled water used was also autoclaved and used specifically in LAMP.Please help out in this.

Thanks
Jasmine
PGIMER

Submitted by Guest (not verified) on

Dear Jasmine
sorry for late reply, i have no time to log in this web:(( however it might possible be contaminated, the autoclaved distilled water is not properly used for this technique, it ll good to use RNAse free water, pls try again and u could not see the precipitation of the reaction in negative control, by naked eyes or/and turbiditymeter.
cheers
Usa

Usa Lek-Uthai's picture
Submitted by Usa Lek-Uthai on

Dear Jasmine,
If u sure that no contaminated LAMP reaction, please again try to run separately and 3-4 blank bands far and make sure that not exceed or over load to each comb, it is possible to have running of band (ladder -like band) because the LAMP product is long and it can be floated (volume overload) and contaminate to nearby comb hole, please try again
cheers
Usa

Usa Lek-Uthai,
Mahidol University,
Bangkok, Thailand

Submitted by Guest (not verified) on

Hi,

I'm a Masters student and my thesis includes LAMP, I designed my primers acording to the rules and with the experience that people on my lab have, but when I use them with any positive sample (DNA extracted from bovine blood samples) there's no amplification, but if I use the outer primers (F3 and B3) in a PCR there is amplification, and if I use these same PCR products as a template for the LAMP there is amplification. My question is: Why does this happen? Is there any kind of reaction inibition? Is the bovine DNA inhibiting the LAMP? I tried pre-denaturating the DNA samples at 95 ºC for 10 minutes but nothing changes.
I would really apreciate any help, thanks

Marcos Santos

Usa Lek-Uthai's picture
Submitted by Usa Lek-Uthai on

Dear Marcos,
Please do new optimization both reagents ' volume (bst polymerase and betaine plus MgSO4 etc should be optimized) and also the condition (temp) should be around 60-68 degree celcious (or a bit over) , if u certainly have a good primer design (BLAST) the F3, B3 primer sequences that can be amplified according to your done. It is possible for PCR rx, pls again check F2, B2 (F1C+ B1C), then if there have no other problems for primers set, optimize again and gel run, any DNA can be amplified by LAMP , not necessary to do pre-nature the samples before LAMP reaction, but u may shock the amplicon with 80 degree celcius for 2 min after end of reaction. (the high temp 95 degree celcius for 10 min will make the DNA post -denaturing shorter sequence than the primer can be looped? I have never do pre-denaturing DNA before the lab. You may email me,(phulu@mahidol.ac.th) As Pooja's now the LAMP problems has been solved:)).
cheers
Usa

Usa Lek-Uthai,
Mahidol University,
Bangkok, Thailand

Submitted by Guest (not verified) on

Dear Usa,

I am a student working on my project on LAMP. I have experience problems same as others that my no template water control is frequently positive and I am desperate about this. I can rule out the possibility of contamination and i have tried to optimize all reagents but there are no significant changes in the water control. Would it be Mia riming of the primers to each other and amplify by the Taq. I have checked for the Blast but the primer blast doesn't allow primers longer than 36bp for blast search. I have tried to lower the dNTP by 10 times than other journals but the problem still exist for the water control. What can I possibly do and try now. Thank you very much for your help and looking forward for your reply.

Regards,
Kate

Submitted by Guest (not verified) on

Dear Kate,
I am a student working with LAMP and was wondering if you ever came up with a solution. Please let me know.
Thanks,
Tyler

Submitted by Guest (not verified) on

Marcos,

I would say the answer is copy number. The assay works when you use mass copy amplicon but not with lower copy pathogen target. Also your total nucleic acid from the target contains millions of extranous bp with secondary structure, etc that compete for your primers. Email me at johndr_tn@yahoo.com, I am also developing a TB specific LAMP assay and perhaps we could talk to exchange ideas and troubleshoot. I am having some issues with my assay right now too.

Thanks,
JOHN

Submitted by Guest (not verified) on

Dear LekuthaiU,

I am trying to apply LAMP for the detection of circovirus in field samples. However instead of clear ladder-like pattern I am getting really big but clear product about 10 kbp long. I am using Bsm Polymerase. Other conditions are identical with mentioned in your post.
After SYBR Green I addition the positive samples give green lightening while the negative control remains orange. (see the picture) http://img560.imageshack.us/img560/3623/lamp.png
Any kind of help or suggestion will be strongly appreciated.

Best wishes,

Greg

Usa Lek-Uthai's picture
Submitted by Usa Lek-Uthai on

Hi Greg
I have never tried the LAMP method in circovirus and also SYBR Green I DNA stained. However , the information u had posted. I could suggest that u might do extract then try to run the gel for the confirmation again. Even this circovirus (??) pentomers, circular genomic, single-stranded DNA 1.8 to 3.8 kb???, in your case, possibly the viruses replicate their genomes via a rolling circle mechanism. A stem loop structure with a conserved nonanucleotide motif may be located at the 5' intergenic region of circovirus genomes and is thought to initiate rolling-cycle replication, the replicon or their replicated products might be packed or big fold. Have u tried to run the gel longer and higher % gel and may sequence it??.
cheers
Usa Lek-Uthai

Usa Lek-Uthai,
Mahidol University,
Bangkok, Thailand

Submitted by Guest (not verified) on

Dear Usa, The problem was in range of temperature since I applied 68 degrees. Now in 62 I got preety good amplification with classical ladder-like pattern. Thank you for your reply. I appreciate this.

Best wishes,

Greg

Submitted by Guest (not verified) on

Dear LekuthaiU,

I'm trying to get the LAMP method working using BSM DNA Polymerase. But I have not got any results so far. I have not been adding Betaine, could that pose a problem. I was wondering if you could guide me through this and on how different BSM DNA polymerase is from BST.

Cheers
Ron!

Usa Lek-Uthai's picture
Submitted by Usa Lek-Uthai on

if this massage 's from Ronnie, I have already replied via email.!! betaine is important for the LAMP reaction, and Bsm polymerase need certainly optimization with temp.
Usa

Usa Lek-Uthai,
Mahidol University,
Bangkok, Thailand

Submitted by Guest (not verified) on

Dear Greg,

I would like to know wheather your LAMP assay with Bsm DNA Polymerase have success or not, because mine haven't. My LAMP was performed for 60 min at temp 60 C in 25 microL of a mixture containing 1 microL of a circular recombinant plasmid containing gene target (i used this plasmid as the template for optimatization assay), 1 microM each of FIP and BIP, 0.2 microM each of F3 and B3, 0.4 mM deoxynucleoside triphosphates, 0.8 M betaine and 1 microL of Bsm DNA polymerase(8U/ microL) in buffer (20 mM Tris–HCl pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, and 0.1% Tween 20) and the reaction was terminated by heating at temp 80 C for 4 min. If yours success, would you like to share me the optimal condition and recipe? Thank you so much

Best Regards,
Issabelmadya

Submitted by Guest (not verified) on

Dear Usa,

I also am having troubles with my no template control (just the reagents + RNase/DNase-free water) getting consitently amplified.

Has anyone solved this problem? It is not contamination of the reagents; perhaps it's the primers amplifying?

Please help with any advice you may have to offer.

Thanks,
Stephanie

Submitted by Guest (not verified) on

Hi Stephanie,

I am consistently having this problem also. I have noticed a few others on this board saying the same thing. I have not yet come across an answer to this, did anyone else solve this problem?....My email is sbasra@uoguelph.ca if anyone is interested in starting a support group email thread for this particular problem.

Thanks,

Simone

Submitted by Guest (not verified) on

Hello Usa et al,

I am also optimising my LAMP assay and finding this amplification with negative controls. I have two sets of negative controls - water and no template at all. Across the range of primer gradients that I have tried, there is amplification in all the negative controls. Is there something else I could optimise? e.g the salt concentrations...

please reply directly via orieroce@yahoo.com thank you.

Usa Lek-Uthai's picture
Submitted by Usa Lek-Uthai on

Hi Stephanie,
it 's possible be contaminated during loading a sample, it wound not be primer amplified. I could suggest that u might try loading the samples and separate or skip 2-3 lanes before a control lane, it is possible as well the amplicon of neighbor lanes will be new reacted with a 2nd primer loading with freely amplification or from contaminated lanes.
Usa

Usa Lek-Uthai,
Mahidol University,
Bangkok, Thailand

Submitted by Guest (not verified) on

I have that experience with my LAMP before, they showed a false-positive when applied without a template. It was suspected that the primers easily formed secondary structures, and 3’ end of primer sequences contained AT rich that could be a complementary to other primers. In general, the LAMP primer set should consist of 50-60% in the case of GC rich and should not be AT rich at 3’ end sequence.

You should avoid AT ricj at 3' end and the stability of RT-LAMP primer is another concern. The primer sequences should be selected based on the change in free energy (∆G) calculated 6 bp from the following end regions which were less than -4 kcal/mol to ensure that the primers did not form complementary and secondary structure to other primers.

-Kate

Submitted by Guest (not verified) on

Hello Stéphanie,

If your negative (water) control is positive, it means that one or more of your reagents (primers included) are contamineted. Maybe your lab environment!
The LoopAmp is very efficient and sensitive. The better way is to prepare your lamp's master mix under a speific DNA/RNA free area (for example: UVP DNA/RNA).

Now, you have to buy fresh new reagents and primers.

Try again!

good luck

Reagards

Emmanuel.

emmanuel.fernandez@cirad.fr

Submitted by Guest (not verified) on

Dear Stephanie,
Did you ever solve this problem? I have run into the same thing.
Thank you,
Tyler

Submitted by gaurvee (not verified) on

Dear Stephanie,
My thesis work is also on LAMP and i am facing the same problem. If you have concluded your study please let me know how did you counter this problem.

Regards,
Gaurvee

Submitted by Guest (not verified) on

Hi, I'm Giana Gomes, I'm new in this group. I work as a researcher worker for the Aquaculture Department from James Cook University-Australia.

I'm starting a project with the Lamp technique. I need to differentiate 2 species of fish using this technique. My question is, how many (minimum) base pairs do I need in the 4 (F3/B3 FIB/BIP) sets of primers to differentiate the 2 species? Is it enough to have just 1 or 2 different base pairs in just 1 or 2 of the 4 sets of primers to give me the specificity for each specie?

Thank you.

Submitted by Guest (not verified) on

I would use restriction enzyme to differentiate them or you have to design two different set LAMP primers. It depends how much close of your species. I have been designed two different virus with the same genus. I careful on FIB and BIP primers.

-Kate

Usa Lek-Uthai's picture
Submitted by Usa Lek-Uthai on

 

Design 4 types of primers (described in detail below) based on the following 6 distinct regions of the target gene: the F3c, F2c and F1c regions at the 3' side and the B1, B2 and B3 regions at the 5' side.


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.
 Main points of primer design



Proper primer design is crucial for performing LAMP amplification.The above primer regions can be determined by using the PrimerExplore (a special software to design LAMP primers) after considering the base composition, GC contents and the formation of secondary structures. Tm value can be obtained by Nearest Neighbor method.
The following is the main points of primer design.
1.Distance between primer regions
· The distance between 5' end of F2 and B2 is considered to be 120-180bp, and the distance between F2 and F3 as well as B2 and B3 is 0-20bp.
· The distance for loop forming regions (5' of F2 to 3' of F1, 5' of B2 to 3' of B1) is 40-60bp.
2.Tm value for primer regions
· About 60-65°C in the case of GC rich and Normal, about 55-60°C for AT rich.
3.The stability of primer end
· The dG calculated on 6bp from the following end regions should be less than -4kcal/mol, 5' end of F1c/B1c and 3' end of F2/B2 as well as F3/B3.
4.GC contents
· About 50-60% in the case of GC rich and Normal, about 40-50% for AT rich.
5.Secondary structure
· Primers should be designed so as not to easily form secondary structures. 3' end sequence should not be AT rich or complementary to other primers.
6.Others
· If the restriction enzyme sites exist on the target sequence, except the primer regions, they can be used to confirm the amplified products.

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

Usa Lek-Uthai,
Mahidol University,
Bangkok, Thailand

Submitted by Guest (not verified) on

 

Hi,

 I designed my primers acording to the rules and with the experience that people

on my lab have, but when I use them with any positive sample,

 if I use the outer primers (F3 and B3) in a PCR there is no amplification,

but  if I use these same  template for the LAMP there is amplification.

My question is: Why does this happen? 
I would really apreciate any help, thanks

Min Wang

Submitted by Guest (not verified) on

Hi Usa Lek-Uthai,

my name is Quang. I'm a student at Hanoi University of Technology-Vietnam, i'm studying LAMP reaction.

in my study process, i wonder whether outer primers (F3 and B3) important for a LAMP reaction? as you see, LAMP reaction can be considered as 2 terms process:

- first term is process to creat the dumbbell structure.Follow the basic principle on LAMP eiken website, in this term, F3 and B3 only play one step. in my opinion, if outer primers absened, then in step 3,  a FIP anneals to the DNA template strand or the new strand synthesised in step 2. Reaction can continue without B3- replace by a BIP primer in step 6, then the dumbbell structure still be formed

- 2nd term is LAMP cycling almost using the dumbbell structure and two inner primers

can you show me this problem? i often check your blog or you can email me with my great thanks.

my email address is: dinhvquang89@gmail.com

Thanks a lot! sorry because of my bad english :D hope you can undersrstand what i say

Usa Lek-Uthai's picture
Submitted by Usa Lek-Uthai on

What is your problem?

 

 

When the target gene (DNA template as example) and the reagents are incubated at a constant temperature between 60-65°C, the following reaction steps proceed:
STEP1
As 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 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.
STEP2
Through 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.
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 because of the displacement by the DNA strand synthesized from the F3 Primer. Then, this released single strand forms a stem-loop structure at the 5' end because of the complementary F1c and F1 regions.
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. The BIP anneals to the DNA strand produced in Step (5). Starting from the 3' end of the BIP, synthesis of complementary DNA takes place. Through this process, the DNA reverts from a loop structure into a linear structure. The B3 Primer anneals to the outside of the BIP and then, through the activity of the DNA polymerase and starting at the 3' end, the DNA synthesized from the BIP is displaced and released as a single strand before DNA synthesis from the B3 Primer.
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). The above process can be understood as producing the starting structure for LAMP cycling.
 Basic principle (8) - (11) (Cycling amplification step)  
A dumbbell-like DNA structure is quickly converted into a stem-loop DNA by self-primed DNA synthesis. FIP anneals to the single stranded region in the stem-loop DNA and primes strand displacement DNA synthesis, releasing the previously synthesized strand. This released single strand forms a stem-loop structure at the 3' end because of complementary B1c and B1 regions. Then, starting from the 3' end of the B1 region, DNA synthesis starts using self-structure as a template, and releases FIP-linked complementary strand (Step (9)). The released single strand then forms a dumbbell-like structure as both ends have complementary F1 - F1c and B1c - B1 regions, respectively (Step (11)). This structure is the 'turn over' structure of the structure formed in Step (8). Similar to the Steps from (8) to (11), structure in Step (11) leads to self-primed DNA synthesis starting from the 3' end of the B1 region. Furthermore, BIP anneals to the B2c region and primes strand displacement DNA synthesis, releasing the B1-primed DNA strand. Accordingly, similar structures to Steps (9) and (10) as well as the same structure as Step (8) are produced. With the structure produced in Step (10), the BIP anneals to the single strand B2c region, and DNA synthesis continues by displacing double stranded DNA sequence. As a result of this process, various sized structures consisting of alternately inverted repeats of the target sequence on the same strand are formed.

 

 

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

Or send your questions to usakitty.lek@gmail.com

 

Usa Lek-Uthai,
Mahidol University,
Bangkok, Thailand

Submitted by Memol (not verified) on

I have problem with false positive. Any solution. I dont know is it possible contamination has come from electrophoresis room in our place?

Submitted by Praveen K S (not verified) on

Dear Scientist,

I have a question and believe to get a prompt reply.

1. What is the necessity of using the Loop Primers when the amplification is happening with F3,B3, FIP & BIP?
2. Would we be viewing only a single band or ladder pattern in the gel electrophoresis from the amplified product without the Loop primers?
3. What would be the base pair of the amplified product over the gel. People say its only one product with single base pair either 200 or 500bp, but my understanding is its different.

Kindly revert back to me asap and thanks in advance.

regards,

Praveen

Submitted by Prashant (not verified) on

Hello,
What is the maximum size of a target region which can be amplified using LAMP?
Is it only 120-180bp as mentioned in the Primer explorer manual v4 or can any length of DNA be amplified?

Usa Lek-Uthai's picture
Submitted by Usa Lek-Uthai on

It 's depend on during the cycling reaction which only the inner primers are used for strand displacement DNA synthesis. The inner primers [forward inner primer (FIP) and the backward inner primer (BIP)], and each contains two distinct sequences corresponding to the sense and antisense sequences of the target DNA. However, the LAMP reaction produced many bands of different sizes from ~300 bp. Production of the bands depended on the presence of the inner primers, the template and DNA polymerase. When the products were analyzed by agarose gel electrophoresis, smeared DNA between bands and at the well may shifted to bands of <10 kb. But remember! less than 1 hr would be the best amplification reaction of this method, so the inner primers that initial reaction to the template and target primers design. I suggest to read more detail in the original paper of Loop-mediated isothermal amplification of DNA...byTsugunori Notomi, Hiroto Okayama,... and Tetsu Hase.
cheers
Usa

Usa Lek-Uthai,
Mahidol University,
Bangkok, Thailand

Submitted by Asyiat&Julia (not verified) on

Hi Usa,
We are trying to develop LAMP assay. However, we haven’t got ladder-like pattern just some 200 b.p. products. LAMP was performed for 90 min in 25 microL of same as yours mixture. We tried to optimize reaction temp (55,60,63,65 C), but nothing changed. No template controls are negative. Can you help us solve this trouble with your experience in LAMP?
Thanks a lot!

Asyiat&Julia

Submitted by Ali (not verified) on

I am using the Bst 2 DNA polymerase for lamp amplification. for setup I use human genomic DNA but I have not see any amplification until now. I tested my primers with PCR and they work properly but they don't anneal in LAMP reaction. I run the temperature gradian but couldn't see amplification in any temperature from 50 to 65. the enzyme buffer which I use has 50 mM KCL (1X) that is more than usual I have seen in articles. can it be the problem? What should I do? How can I test the enzyme to see is it work?

Submitted by farhana (not verified) on

Hi
my thesis is on optimization of LAMP,but i didn't get any result yet.there is no amplification and important point is that im not adding betaine in mixture.Is it due to betaine? betaine is necessary for amplification? kindly reply me
waiting for your answer

Submitted by KARTHIK K (not verified) on

Hi,
What is the condition you are using for your standardization. Give the protocol will try to sort out

Submitted by KARTHIK K (not verified) on

Hi, Give the protocol what u r using. We will try to sort out ur problem

Submitted by Samuel Lee (not verified) on

As LAMP does not require denaturation; using betaine along with a stand separating polymerase allows for amplification. Betaine is often used in PCR for templates with high G/C content to aid in a similar way. I would suggest using betaine.

Submitted by gaurvee (not verified) on

Hello all,
My thesis work is also on LAMP and my work was going just fine for the last one and a half year. But recently i started getting false positives- as the negative control tubes with no template DNA but PCR grade-water also started showing ladder-like pattern in gel electrophoresis. I see a lot of people have also faced this problem. I'd like to know if anybody has found a solution to this.Feedback and help would be appreciated.
Regards,
Gaurvee,
Panjab University,
Chandigarh, India

Submitted by gaurvee (not verified) on

Dear Usa,
My thesis work is also on LAMP and my work was going just fine for the last one and a half year. But recently i started getting false positives- as the negative control tubes with no template DNA but PCR grade-water also started showing ladder-like pattern in gel electrophoresis. I see a lot of people have also faced this problem. I'd like to know if anybody has found a solution to this.Feedback and help would be appreciated.
Regards,
Gaurvee,
Panjab University,
Chandigarh, India

Submitted by Praveen K S on

Dear All,

Any breakthrough w.r.t LAMP Malaria Detection??
I know most of you are trying your level best.
Keep this group active and post as much questions & suggestion as possible so that others can also learn from it.

with regards

Praveen K S,
Senior Lab Technical Officer,
PATH,
Mumbai

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