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LAMP
PRESENTED BY:
DR. CHAYANIKA DAS
Ph.D
VETERINARY MICROBIOLOGY
PCR
• PCR is based on using the ability of DNA polymerase to
synthesize new strand of DNA complementary to the
offered template strand.
• Because DNA polymerase can add nucleotide only onto
pre-existing 3’-OH group, it needs a primer to which it
can add the first nucleotide. This requirement makes it
possible to delineate a specific region of template
sequence that the researcher wants to amplify. At the
end of the PCR reaction, the specific sequence will be
accumulated in billions of copies (amplicons)
LAMP LOOP MEDIATED ISOTHERMAL AMPLIFICATION
• Based on the principle of high strand displacement activity
• Isothermal condition
First described (2000) and initially evaluated for detection of
hepatitis B virus DNA by Notomi et al., EIKEN chemicals Co.Ltd,
Japan
• Detection of Brucella sp. by Chen et al.(2013).
•Detection of Mycobacterium tuberculosis by Kaewphinit et al.
(2013)
SOURCE: Article No. F056E4B44513, African Journal of Biotechnology
Bst DNA polymerase
• Derived from Bacillus stearothermophilus
• Moderately thermostable
• Replicates DNA optimally at 60-65°C
• Can be heat inactivated above 80°C
• Used in isothermal DNA sequencing
• Reverse transcription activity*
Isotherm2G DNA polymerase
• Mesophilic
• Temperature optimum (between 55-70°C)
• High strand displacement activity
• Improved reverse transcription activity
LOOP MEDIATED ISOTHERMAL AMPLIFICATION
Characterized by use of 4 different primers
6 distinct regions of the target gene: F3c,F2c, F1c,B1,B2 & B3
PRINCIPLE
STEP 1
a. Non-cyclic amplification: Generation of stem
loop DNA with dumpbell-shaped structure at
both ends
b. Cyclic amplification: Amplification of dumpbell
shaped DNA with loop primers
NON-CYCLC AMPLIFICATION
STEP 2
STEP 3
STEP 4
STEP 5
STEP 6
STEP 7
STEP 8
DUMPBELL STRUCTURE
CYCLIC AMPLIFICATION
DETECTION OF AMPLICONS
i. Visual detection-
ii. Gel electrophoresis
• Chemical reaction:
Turbidity– Magnesium pyrophosphate
Colour change – Hydroxyl-naphthol blue
• Fluorescence : fluorescent dyes
PCR LAMP
• Isothermal temperature
• Strand displacement activity
•Four primers recognizing 6 distinct sequences
on the target DNA
• Takes less time
•Doesnot require thermocycler
•Detection through gel electrophoresis as well
as through the naked eye (colour change,
turbidity)
• Variable temperature
Denaturation (95°C)
Annealing (50-60°C)
Extension (72°C)
• Denaturation of double stranded forms
•Two primers amplify template DNA
• Time consuming
•Requires thermocycler
•Detection of amplicon through gel
electrophoresis
SOURCE: Article No. F056E4B44513, African Journal of Biotechnology
CONCLUSION
•LAMP is highly specific and efficient nucleic acid amplification
method
• The whole amplification reaction takes place continuously under
isothermal conditions
•Amplification can be done with RNA templates following the
same procedure, simply through the addition of reverse
transcriptase
• The total cost can be reduced, as LAMP doesnot require special
reagents or sophisticated equipments
•An innovative method that can be used for diagnosis of
infectoius diseases, food inspection, environmental testing, etc.
THANK YOU

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LAMP PCR.pptx by Dr. Chayanika Das, Ph.D, Veterinary Microbiology

  • 1. LAMP PRESENTED BY: DR. CHAYANIKA DAS Ph.D VETERINARY MICROBIOLOGY
  • 2. PCR • PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. • Because DNA polymerase can add nucleotide only onto pre-existing 3’-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons)
  • 3. LAMP LOOP MEDIATED ISOTHERMAL AMPLIFICATION • Based on the principle of high strand displacement activity • Isothermal condition First described (2000) and initially evaluated for detection of hepatitis B virus DNA by Notomi et al., EIKEN chemicals Co.Ltd, Japan • Detection of Brucella sp. by Chen et al.(2013). •Detection of Mycobacterium tuberculosis by Kaewphinit et al. (2013) SOURCE: Article No. F056E4B44513, African Journal of Biotechnology
  • 4. Bst DNA polymerase • Derived from Bacillus stearothermophilus • Moderately thermostable • Replicates DNA optimally at 60-65°C • Can be heat inactivated above 80°C • Used in isothermal DNA sequencing • Reverse transcription activity*
  • 5. Isotherm2G DNA polymerase • Mesophilic • Temperature optimum (between 55-70°C) • High strand displacement activity • Improved reverse transcription activity
  • 6. LOOP MEDIATED ISOTHERMAL AMPLIFICATION Characterized by use of 4 different primers
  • 7. 6 distinct regions of the target gene: F3c,F2c, F1c,B1,B2 & B3
  • 8.
  • 9. PRINCIPLE STEP 1 a. Non-cyclic amplification: Generation of stem loop DNA with dumpbell-shaped structure at both ends b. Cyclic amplification: Amplification of dumpbell shaped DNA with loop primers NON-CYCLC AMPLIFICATION
  • 12. STEP 6 STEP 7 STEP 8 DUMPBELL STRUCTURE
  • 14. DETECTION OF AMPLICONS i. Visual detection- ii. Gel electrophoresis • Chemical reaction: Turbidity– Magnesium pyrophosphate Colour change – Hydroxyl-naphthol blue • Fluorescence : fluorescent dyes
  • 15. PCR LAMP • Isothermal temperature • Strand displacement activity •Four primers recognizing 6 distinct sequences on the target DNA • Takes less time •Doesnot require thermocycler •Detection through gel electrophoresis as well as through the naked eye (colour change, turbidity) • Variable temperature Denaturation (95°C) Annealing (50-60°C) Extension (72°C) • Denaturation of double stranded forms •Two primers amplify template DNA • Time consuming •Requires thermocycler •Detection of amplicon through gel electrophoresis SOURCE: Article No. F056E4B44513, African Journal of Biotechnology
  • 16. CONCLUSION •LAMP is highly specific and efficient nucleic acid amplification method • The whole amplification reaction takes place continuously under isothermal conditions •Amplification can be done with RNA templates following the same procedure, simply through the addition of reverse transcriptase • The total cost can be reduced, as LAMP doesnot require special reagents or sophisticated equipments •An innovative method that can be used for diagnosis of infectoius diseases, food inspection, environmental testing, etc.