What is isothermal amplification?
Isothermal amplification is a novel approach to nucleic acid amplification which uses a single temperature incubation removing the need for thermal cycling.
How does LAMP work?
Loop-mediated isothermal DNA amplification (LAMP) is a highly sensitive and specific nucleic acid amplification technique. For more info please click here
What is the difference between LAMP and PCR?
The polymerase chain reaction (PCR) relies upon thermal cycling to achieve DNA strand separation, primer annealing and finally target extension. Loop-mediated isothermal DNA amplification (LAMP) uses a DNA polymerase enzyme with strand displacement activity to achieve strand separation, primer annealing and target extension at a single (isothermal) temperature.
Can I use standard PCR tubes?
Genie® II uses a proprietary tube strip that has a locking cap and maximises both optical and thermal efficiencies. Other tubes cannot be used with the Genie II instrument.
What DNA preparation method is needed?
For highest target specificity and sensitivity we recommend purifying the target DNA/RNA. The strand displacing enzymes offered by OptiGene are less susceptible to inhibitors compared to standard PCR enzymes (e.g. Taq) and as such may need little/no template purification.
How fast should I expect a result?
Target amplification will vary depending on a number of factors including primer design (we recommend LAMPDesigner®), template purity and template quantity. Typically the ISO-001 mastermix can achieve positive results between 5 and 30 minutes.
How do I design primers for LAMP?
LAMP requires 6 primers with 8 specificities, we supply a LAMP designer software to offer an easy alternative and allow rapid and easy design. For more info on the software, please click here
Do my primers need any special purification protocols?
No. Standard desalted oligo primers are suitable.
How do I confirm a positive result?
LAMP reactions produce amplicons with a specific sequence unique to that target. As such the amplicon will produce a unique annealing temperature (similar to high resolution melting) that can confirm amplification of the correct target. In the same way, false positive and negative results can be easily be distinguished.
Do I have to use LAMP?
There are many Isothermal amplification methods and nearly all can be used on the Genie® II system. RPA, NASBA, SMAP and BART have all been run successfully.
How specific is LAMP?
LAMP requires 6 unique primers annealing up to 8 different sites on each target. As such, LAMP offers a highly specific amplification technique. In comparison, PCR requires only 2 sites to yield amplification.
Is LAMP affected by common PCR inhibitors?
The strand displacing DNA polymerases employed in LAMP show greater tolerance to common inhibitors (Blood, soil etc.) when compared to PCR enzymes. For optimum specificity and sensitivity we recommend purifying target DNA and RNA before use.
What advantages does LAMP offer over PCR?
Isothermal nucleic amplification techniques (e.g. LAMP) do not require the thermal cycling of PCR and as such enable smaller, lighter and cheaper instrumentation. LAMP and PCR offer similar specificities and sensitivities. LAMP enzymes and reagents are more robust than PCR mixes.
What does the Genie®II do?
The Genie®II is specifically designed to run any isothermal amplification method that employs target detection by fluorescence measurement. Genie® II will detect all dyes that can be excited from a blue light source and with an emission above 510 nm. Further uses include enzyme kinetic analysis and protein denaturation analysis using fluorescent dyes.
Who can use a Genie?
The Genie instrument is designed to be used by anyone with basic training. Simple reaction set up with easy to use touchscreen operation facilitates molecular detection for all.
Does the Genie® need to be connected to a computer?
The Genie®II is a stand-alone instruments and as such do not require a computer.
How long will Genie® II run from Battery?
Genie® II will run for a full working day from the internal battery.
How long does Genie® II take to charge the battery?
From empty Genie® II will charge fully in less than 3 hours.
What other materials do I need to use the Genie®II?
Custom Genie®II 8-well reaction tubes. Pipettes and tips. Assay reagents (e.g. LAMP: ISO-001 mastermix, primers, template DNA/RNA, sterile water).
Can I use my own reagents on the Genie®?
The Genie®II is an open platform suitable for all chemistries facilitating fluorescent detection excited from a blue light source and with an emission above 510 nm. For optimal performance we recommend using ISO-001 mastermix for the fastest possible LAMP reactions.
Can I use intercalating dyes for real-time monitoring with Genie®II?
The Genie®II is specifically designed to run any methods that employing fluorescence measurement. Genie® II will detect all dyes that can be excited from a blue light source and with an emission above 510 nm.
Can I use my existing PCR primers/probes?
No. LAMP requires 6 specific primers to enable a successful amplification reaction. Please see LAMPDesigner for more information.
Can I run multiplex LAMP reactions?
Yes. Caution must be taken with primer design.
How much of my primers do I have to use?
We recommend 1.6µM FIP and BIP primers, 0.4µM F3 and B3 primers, 0.8µM LoopF and LoopB primers per reaction.
Can I use the two blocks independently?
Each block can be used independently, Different protocols can be run on the two blocks and they can be started and end at different times. When optimization of a new isothermal assay is required it is possible to run with a thermal gradient along a block so that each well is at a different temperature.
What temperature range can Genie® II cover?
The temperature control system within Genie® II works over a temperature range of 40°C to 99°C.
How do I get software/firmware updates?
Software and firmware updates are available from the OptiGene website and can easily be applied using the update function on Genie® II.
What dyes can I use with Genie® II?
Genie® II will detect all dyes that can be excited from a blue light source and with an emission above 510 nm.
What is an annealing curve?
Performing an annealing step, rather than a melt, on the finished amplification product results in a single product peak from which a Ta value can be extracted. Similar to a melt curve, the annealing Ta value is unique to the sequence amplified, confirming amplification of the correct product.
Do I need to do a pre-heat before amplification?
It is not mandatory to do a 95°C preheat prior to amplification but this can be included in the thermal protocol if required.
What is in the mastermix?
OptiGene’s proprietary enzyme along with Magnesium and buffers in the correct ratios to allow the user to quickly produce the best results from their Isothermal assay.
What are the differences between the mastermixes?
Isothermal Mastermix – ISO-001: This isothermal amplification mix allows fluorescence detection of the product on the Genie® II platform but may also be used on generic qPCR instrumentation. An anneal curve can then be generated to confirm the product. This eliminates the need for gel electrophoresis or turbidity detection and allows for a closed-tube system.
Isothermal Mastermix, no dye – ISO-001nd: This isothermal amplification mix allows the addition of alternative dyes or probes for use on a Genie® II. It could alternately be used for end-point gel electrophoresis detection where contamination is not an issue.
Isothermal Mastermix, no dye, no pyrophosphatase – ISO-001nd-npy: This isothermal amplification mix is suitable for turbidimetric detection. All OptiGene Isothermal Mastermixes are fully licensed with the Eiken Chemical Company for use in LAMP reactions. OptiGene Isothermal Mastermix can be used with RCA, SMAP and many other amplification technologies.
Do I need to run a control?
We recommend running control reactions (positive and negative) for all laboratory techniques.
What does a positive result look like?
Positive target amplification can be visualised by an increase in fluorescence. Only if your target is present will dsDNA be generated allowing a dsDNA intercalating dye to fluoresce. A positive amplification plot will show an ‘S’ shaped sigmoid curve reflecting the increase in fluorescence detected.
Can I use amplification reactions for the quantification of template?
Unlike PCR, LAMP reactions do not amplify DNA exponentially. As such, target concentrations can be estimated but not truly quantified using LAMP.
Can I perform a fluorescence end-point analysis of the amplification reaction?
Yes. Reaction products can be further confirmed by visualisation on an agarose gel by the addition of a fluorescently labelled intercalating dye (e.g. ethidium bromide or SYBR green). We do not recommend end-point visualisation due to the risk of contamination from opening the reaction tube which may contain >20mg amplicon!
What temperature should I run LAMP reactions?
The exact temperature will depend on your LAMP primer set but primers should be designed to optimally anneal at 65°C. The ISO-001 mastermix contains an enzyme with optimal activity at 65°C. The ability of the Genie®II to run a temperature gradient allows for easy assay optimisation.
Can I use LAMP amplicons for TA cloning?
Yes. LAMP amplicons can be TA cloned if desired for further sequence verification.
Can I amplify RNA?
Yes. RT-LAMP is achieved through the addition of a separate reverse transcriptase enzyme. The ISO-001 mastermix shows inherent RT-LAMP activity and does not require the addition of a separate RT enzyme.
How can I clean/decontaminate the Genie®II?
Carefully wipe down the exterior of the machine with a damp cloth with 5% bleach