MolBIO - Isothermal Amplification Techniques

Nucleic Acid Sequence-Based Amplification (NASBA)

Reaction: Isothermal Amplification and Retroviral RNA Replication

RT Enzyme Activity: RNase H added to remove RNA from cDNA without heat-denaturation

Transcription-mediated Amplification (TMA)

Reaction: Isothermal Amplification and Retroviral RNA Replication

RT Enzyme Activity: RT Enzyme degrades initial RNA template after synthesis to cDNA

Signal-Mediated Amplification of RNA Technology (SMART)

Formation of a 3 way junction structure where 2 probes annealed to each other in the presence of a specific target

Strand Displacement Amplification (SDA)

Uses Restriction Endonucleases and Exonuclease-deficient strand displacing DNA Polymerase for DNA Replication

After denaturation:

• Extension of SDA Primers with restriction sites

• Displacement by extension of flanking bumper primers

• Product: Double-stranded target sequence flanked by nickable restriction sites

Rolling Circle Amplification

Strand-displacing DNA Polymerase

• amplify circular DNA template at low temperatures

• Product: DNA with tandem repeats

The product is used as a template producing long ssDNA with repeated sequences of the original target

Loop-Mediated Isothermal Amplification

• Rapid testing

• Reverse Transcriptase added for RNA

• Products:

  • Stem Loop DNA structures with several inverted repeats

  • Cauliflower-like structures with multiple loops

• Byproduct: Pyrophosphate (White precipitate)

  • Due to synthesis of large amounts of DNA in a short time

• To detect reactions:

  • Observing the precipitate

  • Quantitate: Turbidity change

Amplification

PCR

Cycles through 3 temperature steps:

• Denaturation at 95°C,

• Primer annealing at ~60°C,

• Polymerization at 72°C

LAMP

• Works under a constant temperature usually between 60-65°C

Denaturation

PCR

• High temperature required for separation of strands, enabling primer binding

LAMP

• Denaturation step is performed by strand displacing polymerase

Equipment

PCR

• Thermocycler

LAMP

• Doesn’t require dedicated thermocycler; can use a simple water bath

Reaction Time

PCR

• At least 90 minutes

LAMP

• Results are typically ready in less than 30 minutes

Sensitivity

PCR

• Detects targets starting at nanogram levels

LAMP

• Detects targets starting at femtogram levels

Specificity

PCR

• Requires careful primer design to avoid primer dimer or non-specific amplification

LAMP

• Tolerates (works well with) multiple primer combinations for greater specificity

DNA Template preparation

PCR

• Requires purification or special handling for high sensitivity and specificity

LAMP

• Tolerates inherent impurities and inhibitors common to field samples with highest sensitivity and specificity

DNA Visualization

PCR

• DNA visualization only possible after gel electrophoresis

LAMP

• Immediate visualization of DNA by colorimetry/visual turbidity

Isothermal multiple displacement amplification

Strand displacement replication of the nucleic acid by multiple primers

2 primer sets used

• Right set: complementary to the strand to be amplified

• Left set: complementary to the opposite strand

Requires specific DNA polymerase

Single Primer Isothermal Amplification

Uses a single chimeric primer for amplification of DNA and RNA

Composed of

• Deoxyribonucleotide at the 3’ end

• Ribonucleotide at the 5’ end

• RNase H

• DNA Polymerase

Helicase Dependent Amplification

Based on the DNA replication fork

DNA Helicase - dsDNA

• Removing 95C denaturation step in PCR

• ssDNA protected by SSB Protein

Newly synthesized dsDNA are used as substrates for the next round

Steps of LAMP

1. Annealing of Primers

2. Complementary DNA synthesis

3. Annealing of F3 Primers at F3c region

4. dsDNA formed

5. Release of FIP-linked complementary strand

6. BIP DNA synthesis

7. dsDNA produced

8. Stem-loop structure

Steps of LAMP

1. AoP

2. CDNAs

3. F3 and F3c

4. dsDNA

5. rFIP

6. BIP DNA

7. dsDNA

8. SLS