Loop-Mediated Isothermal Amplification (LAMP) – Key Vocabulary

Background & Rationale

• Nucleic‐acid amplification underpins modern life-science and medical diagnostics.

  • Applications span infectious‐disease detection, genetic-disorder screening, trait identification, etc.

• Conventional and contemporary methods reviewed in the paper and their limitations:

  • Polymerase Chain Reaction (PCR)

  • Requires repeated thermal cycling (denaturation, annealing, extension).

  • Needs precision equipment ⇒ limited point-of-care use.

  • Nucleic Acid Sequence–Based Amplification (NASBA) & Self-Sustained Sequence Replication (3SR)

  • Isothermal (≈40\,^{\circ}\mathrm{C}) but lower specificity because of low temperature.

  • Strand Displacement Amplification (SDA)

  • Uses four primers, restriction enzymes, modified nucleotides.

  • Background from non-target digestion; reagent cost is high.

• Residual “co-amplification” of irrelevant sequences is a universal problem, especially in diagnostics.

• Aim of the study: introduce Loop-Mediated Isothermal Amplification (LAMP) to solve specificity, speed, and equipment‐dependency issues.

Core Principle of LAMP

• Amplifies DNA at a single temperature (optimal 60–65\,^{\circ}\mathrm{C}).

• Requires:

  • A DNA polymerase with strong strand-displacement activity (e.g., Bst Large Fragment, BcaBEST).

  • Four primers recognising six sequences on the target:

  • Inner primers: Forward Inner Primer (FIP) & Backward Inner Primer (BIP).

    • Each contains two regions (sense & antisense) joined by a TTTT spacer → enables self-priming.

  • Outer primers: F3 & B3.

    • Shorter; used at \tfrac{1}{4}–\tfrac{1}{10} the concentration of inner primers.

• Mechanistic stages (see Fig. 1 of paper):

  1. Denature target, rapid cooling, primer binding.

  2. FIP (or BIP) starts synthesis on F2c (or B2c).

  3. F3 (or B3) initiates strand displacement ⇒ releases single strand with a loop at one end (dumb-bell precursor).

  4. Complementary events at opposite end generate a full dumb-bell that self-primes to a stem–loop DNA (structure 7).

  5. Cyclic amplification:

  • Inner primer binds loop, extends & displaces.

  • Each half-cycle triples target copies ⇒ \text{copies}{t+0.5}\approx3\times\text{copies}t.

  1. Products accumulate to 10^9 copies in <60\,\text{min}.

• Final molecular species:

  • Stem–loop DNAs with tandem inverted repeats.

  • “Cauliflower” structures bearing multiple loops.

• Specificity logic:

  • Initial recognition by 6 sequences; cyclic phase by 4 sequences ⇒ improbability of non-specific co-amplification.

Reaction Formulation (Standard 25 µL)

• 0.8\,\mu\text{M} each of FIP & BIP.

• 0.2\,\mu\text{M} each of F3 & B3.

• 400\,\mu\text{M} each dNTP.

• 1\,\text{M} betaine.

• Buffer: 20\,\text{mM} Tris–HCl (pH 8.8), 10\,\text{mM} KCl, 10\,\text{mM} (NH4)2SO4, 4\,\text{mM} MgSO4, 0.1\,\% Triton X-100.

• Thermal profile:

  1. 95\,^{\circ}\mathrm{C} × 5 min (denature).

  2. Ice chill.

  3. Add 8 U Bst polymerase.

  4. 65\,^{\circ}\mathrm{C} × 60 min (amplify).

  5. 80\,^{\circ}\mathrm{C} × 10 min (terminate).

Design Rules for Efficient LAMP

• Melting temperature (T_m) windows:

  • F2 & B2 regions: 60–65\,^{\circ}\mathrm{C} (matches enzyme optimum).

  • F1c & B1c: slightly higher than F2/B2 to ensure prompt loop formation.

  • Outer primers (F3/B3): lower T_m than inner segments.

• Loop length between F2c–F1c or B2c–B1c ≥ 40 nt.

• Optimal target size: 130–200\,\text{bp}; acceptable <300\,\text{bp} (longer targets slow strand displacement).

• Chemical enhancers: betaine or L-proline (0.5–1.5 M) destabilise base stacking, boosting speed & specificity.

Experimental Demonstrations

Model System: M13mp18 DNA (600 copies)

• Primers designed (explicit sequences provided; see Materials section).

• Observations:

  • Agarose gel: ladder-like bands (≈300 bp → well) absent without inner primers, template, or enzyme.

  • Alkaline gel: bulk of slow-migrating DNA resolves <10 kb ⇒ confirms single-strand looped structures.

  • Restriction mapping:

  • BamHI ⇒ \approx100 & 230\,\text{bp}.

  • PstI ⇒ \approx140 & 200\,\text{bp}.

  • PvuII ⇒ \approx240, 320, 350\,\text{bp}.

  • Southern blots with probes M13-281, M13-333, BIP validate identity/location of fragments.

  • Cloning & sequencing corroborate predicted inverted-repeat architecture.

Sensitivity Study: Hepatitis B Virus (HBV) Target

• HBV DNA inserted in pBR322; LAMP at 60\,^{\circ}\mathrm{C}.

• Detection threshold: 6\,\text{copies} in 45 min (fluorescence via SYBR Green I).

• Primer dependency:

  • Removal of any single primer or replacing FIP/BIP with loop-deficient analogs ⇒ no amplification.

• Robustness to background DNA:

  • Presence of 100\,\text{ng} human genomic DNA did not inhibit HBV detection or cause false positives.

  • Under identical conditions single PCR failed; nested PCR was required—highlighting LAMP’s superiority.

Reverse-Transcription LAMP (RT-LAMP): Prostate-Specific Antigen (PSA) mRNA

• Mixed samples: 1 LNCaP cell (PSA⁺) + 10^6 K562 cells (PSA⁻).

• Reaction components added simultaneously on ice; incubation 65\,^{\circ}\mathrm{C} × 45 min.

• Detection:

  • Bands present only when both ReverTra Ace (RTase) & Bst polymerase supplied.

  • Sau3AI digestion yielded expected fragments, verifying product fidelity.

Optimisation & Enzyme Choices

• Bst polymerase & BcaBEST: best for ≤10^{-23}\,\text{mol} template.

• Z-Taq workable if enzyme must survive initial denaturation.

Comparative Advantages of LAMP

• Isothermal amplification—only a water bath or heat block needed ⇒ ideal for point-of-care, field, low-resource settings.

• Speed: 30–60 min for ≈10^9-fold amplification.

• High analytical specificity from multi-site recognition (6 → 4 sites per cycle).

• High analytical sensitivity (few-copy detection comparable to or better than PCR).

• Product geometry (multiple loops) opens avenues for simple detection (e.g., turbidity, fluorescence, lateral-flow via multivalent binding).

• Compatible with RNA templates when combined with RTase.

• Lower inhibition by non-target DNA compared with single PCR.

Practical & Ethical Considerations

• Clinical diagnostics: rapid detection of pathogens (HBV, MTB, SARS-CoV-2 analogues), genetic markers.

• Public-health impact: enables testing in remote clinics without advanced thermocyclers.

• Quality-control imperative: multi-primer requirement reduces false positives but demands careful primer design to avoid cross-reactivity.

• Cost considerations: avoids expensive modified nucleotides & restriction enzymes required in SDA.

• Ethical deployment: faster infectious-disease diagnosis can improve patient care but also necessitates data privacy, informed consent, and equitable access.

Key Numerical & Chemical Highlights

• Copy-number gain: up to 10^9 in <60\,\text{min}.

• Primer concentrations: inner 0.8\,\mu\text{M}; outer 0.2\,\mu\text{M}.

• Betaine optimum: 0.5–1.5\,\text{M}.

• Temperature window: 60–65\,^{\circ}\mathrm{C}.

• Loop length criterion: ≥40\,\text{nt}.

• Target length ideal: 130–200\,\text{bp}; maximal practical <300\,\text{bp}.

Workflow Summary (Bench Top)

1. Design 4 primers following Tm/loop guidelines.

2. Prepare reaction cocktail (may add RTase for RNA diagnostics).

3. Denature sample briefly; cool; add strand-displacement polymerase.

4. Incubate isothermally; monitor in real time (fluorescence/turbidity) or endpoint (gel electrophoresis, color change).

5. Confirm specificity via restriction digest or sequence-specific probes if needed.

Concluding Statements

• LAMP achieves the trifecta of speed, simplicity and specificity; poised to replace or complement PCR in many settings.

• Continuous refinement (e.g., visual dyes, microfluidic integration, CRISPR-LAMP hybrids) is expanding its diagnostic relevance.

• Researchers and clinicians should weigh primer-design complexity against the operational advantages for their particular application.