Isothermal amplification
Loop-mediated isothermal amplification: four to six primers and a strand-displacing polymerase at around 65 °C build a self-priming dumbbell intermediate and then amplify it into long concatemers of the target, making micrograms of DNA that can be read by fluorescence, turbidity or a colour change you can see.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
LAMP
Loop-mediated isothermal amplification: four to six primers and a strand-displacing polymerase at around 65 °C build a self-priming dumbbell intermediate and then amplify it into long concatemers of the target, making micrograms of DNA that can be read by fluorescence, turbidity or a colour change you can see.
How it differs from the standard method
- Six primers rather than four: the loop primers LF and LB roughly halve the time to positive, and roughly double the primer-driven background.
- Needs no denaturation step, but the inner primers' two-part design means an assay stands or falls on its primer set.
- Product is a ladder of concatemers, not a single band, so a gel cannot distinguish a real positive from a primer artefact.
- The colorimetric version needs a nearly unbuffered mix, so a buffered sample or too much sample volume stops the colour changing at all.
- Reverse transcription can be included in the same tube at the same temperature (RT-LAMP), which is how RNA targets are detected in one step.
1Amplifying without a thermocycler
PCR needs 95 °C because that is how the two strands of a duplex are separated so primers can get in. Isothermal methods solve the same problem a different way: they use an enzyme that can open or displace DNA at its working temperature, so the reaction never has to be heated and cooled at all.
LAMP
Four to six primers build a self-priming dumbbell, then amplify it into a ladder of concatemers. Fast, very sensitive, tolerant of crude sample, and prone to amplifying itself.
RPA
A recombinase pushes two primers into intact duplex DNA with no heat at all. Finished in under ten minutes, at body temperature.
RCA
One highly processive polymerase goes round and round a circular template, making a tandem repeat of it. Linear unless you add a second primer.
What it costs
No cycles means no Cq. You get a time-to-positive, which moves only a couple of minutes per ten-fold change in template.
2How LAMP actually works
LAMP is the one worth understanding in detail, because its primer design is the whole technique. Two inner primers, FIP and BIP, each carry two sequences: one that binds the target and one that is the reverse complement of a site further in.
3A time-to-positive is not a Cq
If you watch an isothermal reaction with a dye, you get a curve that rises and plateaus just like a qPCR trace, but with minutes on the x-axis instead of cycles, and the number you report is the time the signal crossed a threshold. It is still a logarithmic read on how much template you started with (a reaction that starts from ten times more target crosses earlier by ln(10)/k, where k is the exponential rate) but the rate is high, so ten-fold costs only a couple of minutes, and that interval is not stable between runs.
- Strand displacement
- A polymerase continuing through a duplex and pushing the existing strand off, rather than stopping. It does the job that 95 °C does in PCR.
- Dumbbell structure
- The self-priming LAMP intermediate: a strand with a stem-loop at each end, formed by the inner primers' two-part design.
- Loop primers (LF/LB)
- Two extra primers that bind the dumbbell's single-stranded loops, roughly halving the time to positive, and raising the background.
- Time-to-positive
- The minute at which the signal crossed its threshold. The isothermal equivalent of a Cq, and a much coarser one.
- Recombinase (uvsX)
- In RPA, the protein that loads onto a primer and invades intact duplex DNA to find its site, so no denaturation step is needed.
- Processivity
- How far a polymerase goes before letting go. phi29's is tens of kilobases, which is what makes a rolling-circle product one long molecule.
- Padlock probe
- A linear oligo whose two ends are brought together on a target and ligated into a circle. Only a circle can be rolled, so ligation is the detection event.
- Carry-over contamination
- Product from a previous reaction seeding a new one. The defining failure mode of isothermal assays, because the product is abundant and the formats are open.
4Reading the tube without an instrument
Most of the appeal of isothermal amplification is that you can read it by eye, and the different read-outs are simply different amounts of product. A real-time dye sees about a nanogram of double-stranded DNA, so it reports first.
5Why contamination is the defining problem
A finished LAMP reaction holds something like 10¹² copies of the target in 25 µL. Open that tube anywhere near where reactions are set up and you have aerosolised more template than any sample will ever contain; a tube rack that held one carries enough on it to seed the next batch.
Key terms
Practise these as flashcards →Held at one temperature throughout. There is no cycling, so product accumulates in time rather than in cycles.
The thermostable, strand-displacing polymerase used in LAMP, working around 60–65 °C and starting to denature above about 68 °C.
A highly processive, mesophilic polymerase from a bacteriophage, used for rolling-circle amplification at about 30 °C and inactivated above 37 °C.
Adding a primer complementary to the rolling-circle product so new strands start on it, turning a linear reaction into an exponential one with a bulk signal.
Read-out using a pH indicator in a weakly buffered mix: polymerisation releases protons, the pH falls and phenol red turns from pink to yellow.
Magnesium pyrophosphate, a by-product of nucleotide incorporation, precipitating enough to cloud the tube visibly.
The RPA-specific fluorescent probe, cleaved by an exonuclease only when hybridised to the amplicon, which makes a real-time RPA read sequence-specific.
Other isothermal chemistries built on the same idea with different enzymes (a helicase, a nicking enzyme, or an RNA-based cycle) used where their particular format suits.
When to use it
- You need an answer where there is no thermocycler: a field survey, a clinic without a laboratory, a bedside or a classroom.
- You need a result in minutes rather than an hour, and a yes/no or a rough amount is enough.
- Your sample is crude. Bst and the RPA enzymes tolerate far more heat-lysate, soil extract or plant macerate than Taq does, which can remove the extraction step entirely.
- You want a single-molecule read-out in situ: rolling-circle amplification on a ligated padlock probe makes one bright spot per detected molecule, which is the basis of in-situ and proximity-ligation detection.
- You do not need quantification. If the question is how much, this is the wrong technique.
Compared with alternatives
Needs a thermocycler but gives a single defined amplicon, far better specificity from two primers, and a product you can clone or sequence directly.
Prefer it whenever the answer is a number: a Cq against a standard curve is a real measurement, where a time-to-positive moves only a couple of minutes per ten-fold.
Prefer it for an absolute count or a rare variant. Partition-and-count has the precision that isothermal kinetics can never give.
The usual way to look at the product, but a LAMP ladder looks much the same whether it came from the target or from the primers, so a gel is a poor specificity check here.
Uses rolling-circle amplification as its amplification step: a ligated circle rolled into a long concatemer becomes one countable fluorescent spot per detected interaction.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Loop-mediated isothermal amplification: four to six primers and a strand-displacing polymerase at around 65 °C build a self-priming dumbbell intermediate and then amplify it into long concatemers of the target, making micrograms of DNA that can be read by fluorescence, turbidity or a colour change you can see.
- Six primers rather than four: the loop primers LF and LB roughly halve the time to positive, and roughly double the primer-driven background.
- Needs no denaturation step, but the inner primers' two-part design means an assay stands or falls on its primer set.
- Product is a ladder of concatemers, not a single band, so a gel cannot distinguish a real positive from a primer artefact.
- The colorimetric version needs a nearly unbuffered mix, so a buffered sample or too much sample volume stops the colour changing at all.
- Reverse transcription can be included in the same tube at the same temperature (RT-LAMP), which is how RNA targets are detected in one step.