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Loop-mediated isothermal             amplification (LAMP) of DNA                         Rokshana ParvinINSTITUT FÜR VIROL...
Nucleic acid amplification is one of the most valuable tools in       virtually all life science fields             PCR; ...
INSTITUT FÜR VIROLOGIE
INSTITUT FÜR VIROLOGIE
http://loopamp.eiken.co.jp/e/lamp/index.htmlINSTITUT FÜR VIROLOGIE
INSTITUT FÜR VIROLOGIE
Loop mediated isothermal amplification (LAMP) is a         single tube technique for the amplification of DNA offering    ...
Characteristics  ─ no need for a step to denature double stranded into a single stranded form.  ─ The whole amplification ...
Design of primers   Design 4 types of primers based on the following 6 distinct regions of the target   gene:  At the 3 si...
T                               CA                                                                 C                      ...
Reaction mixture for LAMP                         0.8 µM each BIP&FIP                          0.2 µM each F3&B3          ...
Betaine                                                    Reduce base stacking,                                         ...
Mechanism or Basic principle 1. Initial step: starting material producing step Double stranded DNA is in the condition of ...
STEP3 The F3 Primer anneals to the F3c region, outside of FIP, on the target DNA and initiates strand displacement DNA syn...
STEP6  This single strand DNA in Step (5) serves as a  template for BIP-initiated DNA synthesis and  subsequent B3-primed ...
2. Cycling amplification step  FIP anneals to stem- loop  primes strand displacement  Generate one stem-loop and  addition...
3. Elongation and recycling step    The final products are mixture of stem-    loop DNAs with stem lengths and    cauliflo...
Mechanism or Basic principle- Video http://www.youtube.com/watch?v=5Wi-kkSFy48           http://loopamp.eiken.co.jp/e/lamp...
Use of loop primer The Loop Primers containing sequences complementary to the single stranded loop region (either between ...
Analysis of product      1. Gel elctrophoresis: 2-3% agarose gel stained with EB / SYBR green or         Calcein      2. N...
INSTITUT FÜR VIROLOGIE
A) The positive PCR reactions show a 273 bp amplicon while          A) LAMP produces a characteristic ladder of multiple b...
Visual detection of sample by LAMP from                         positive and negative samples.                         (1)...
Uses and benefits         Simple screening assay in the field or at the point of care by clinicians         Low cost    ...
INSTITUT FÜR VIROLOGIE
INSTITUT FÜR VIROLOGIE
INSTITUT FÜR VIROLOGIE
Thank you so much for                   your kind attentionINSTITUT FÜR VIROLOGIE
In the DNA amplification process by DNA polymerase, pyrophosphate ions are produced as a    by-product from the reaction s...
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Journal club 11 06-12

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Journal club 11 06-12

  1. 1. Loop-mediated isothermal amplification (LAMP) of DNA Rokshana ParvinINSTITUT FÜR VIROLOGIE
  2. 2. 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 amplificationINSTITUT FÜR VIROLOGIE
  3. 3. INSTITUT FÜR VIROLOGIE
  4. 4. INSTITUT FÜR VIROLOGIE
  5. 5. http://loopamp.eiken.co.jp/e/lamp/index.htmlINSTITUT FÜR VIROLOGIE
  6. 6. INSTITUT FÜR VIROLOGIE
  7. 7. Loop mediated isothermal amplification (LAMP) is a single tube technique for the amplification of DNA offering rapid, accurate, and cost-effective diagnosis of infectious diseasesINSTITUT FÜR VIROLOGIE
  8. 8. Characteristics ─ 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 equipments (Thermal cycler). ─ Amplification also can be done with RNA templates following the same procedure, simply through the addition of reverse transcriptase.INSTITUT FÜR VIROLOGIE
  9. 9. Design of primers Design 4 types of primers based on the following 6 distinct regions of the target gene: At the 3 side: F3c, F2c and F1c regions F3c F2c F1c Target DNA 3 5 5 3 F3 F2 F1 At the 5 side: B1, B2 and B3 regions Target DNA B1 B2 B3 3 5 5 3 B1c B2c B3cINSTITUT FÜR VIROLOGIE
  10. 10. T CA C G T FIP Forward Inner Primer (FIP) consists of the F2 A T G region (at the 3 end) that is complementary to the F2c region, and the same sequence as the GA C F1c region at the 5 end. F3 Forward Outer Primer consists of the F3 region Primer 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 Backward Outer Primer consists of the B3 Primer region that is complementary to the B3c region. http://loopamp.eiken.co.jp/e/lamp/primer.htmlINSTITUT FÜR VIROLOGIE
  11. 11. Reaction mixture for LAMP 0.8 µM each BIP&FIP 0.2 µM each F3&B3 400 µM dNTPs 1M betaine 25 µl 20 mM Tris-HCL Reaction Mixture 10 mM KCL 60-65 C for 1 h 10 mM (NH4) 2 SO4 4 mM MgSO4 0.1% Triton X-100 Bst DNA polymerase Target DNA Terminate by 80- 90 C for 5-10 mINSTITUT FÜR VIROLOGIE
  12. 12. Betaine  Reduce base stacking,  Stimulated the overwall rate of the reaction  Increased target selectivity with a significant reduction of irrelevent sequences N,N,N-trimethylglycine Bst DNA polymeraseBst polymerase is a thermostable DNA Pol I that wasisolated in 1968 (Stenesh 1968) from the thermophilicbacterium Bacillus stearothermophilus (Bst), whichproliferates between 39 and 70 C. Bst Pol I is active atan optimal temperature of 65 C, and is inactivated after15 min incubation at 75 C.INSTITUT FÜR VIROLOGIE
  13. 13. Mechanism or Basic principle 1. Initial step: starting material producing step 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.STEP1Inner primer FIP anneal to F2c in the target DNASTEP2Through the activity of DNA polymerase with stranddisplacement activity,initates complementary strandsynthesisINSTITUT FÜR VIROLOGIE
  14. 14. 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 regionsINSTITUT FÜR VIROLOGIE
  15. 15. 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. 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).INSTITUT FÜR VIROLOGIE
  16. 16. 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 cycleINSTITUT FÜR VIROLOGIE
  17. 17. 3. Elongation and recycling step The final products are mixture of stem- loop DNAs with stem lengths and cauliflower - like structures with multiple copiesINSTITUT FÜR VIROLOGIE
  18. 18. Mechanism or Basic principle- Video http://www.youtube.com/watch?v=5Wi-kkSFy48 http://loopamp.eiken.co.jp/e/lamp/anim.htmlINSTITUT FÜR VIROLOGIE
  19. 19. Use of loop primer 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. 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.INSTITUT FÜR VIROLOGIE
  20. 20. 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.INSTITUT FÜR VIROLOGIE
  21. 21. INSTITUT FÜR VIROLOGIE
  22. 22. 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.INSTITUT FÜR VIROLOGIE
  23. 23. 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.INSTITUT FÜR VIROLOGIE
  24. 24. Uses and benefits  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 virusesINSTITUT FÜR VIROLOGIE
  25. 25. INSTITUT FÜR VIROLOGIE
  26. 26. INSTITUT FÜR VIROLOGIE
  27. 27. INSTITUT FÜR VIROLOGIE
  28. 28. Thank you so much for your kind attentionINSTITUT FÜR VIROLOGIE
  29. 29. 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.INSTITUT FÜR VIROLOGIE

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