Nucleic Acid Target Amplification Techniques: Nucleic Acid Target Amplification Techniques

What are isothermal target amplification methods?

Miniaturized amplification systems that amplify nucleic acids at a constant temperature without thermal cycling.

How do isothermal methods differ from PCR?

PCR requires repetitive thermal cycling, while isothermal methods amplify nucleic acids continuously at one temperature.

Name some examples of isothermal amplification methods.

• Transcription Mediated Amplification (TMA)

• Nucleic Acid Sequence-Based Amplification (NASBA) – similar to TMA

• Strand Displacement Amplification (SDA)

• Multiple Displacement Amplification (MDA)

• Loop-Mediated Isothermal Amplification (LAMP)

• Helicase-Dependent Amplification (HDA)

• Rolling Circle Amplification (RCA)

• Recombinase Polymerase Amplification (RPA)

What is a key feature of Transcription Mediated Amplification (TMA)

Produces 100–1000 copies per cycle and targets RNA, which is more abundant per cell compared to DNA (2 copies/cell).

What are the main advantages of isothermal amplification?

• Fast turnaround time (TAT)

• Portable devices suitable for rural areas

• Miniaturized and low power consumption

• Battery-operated detection systems

• Wide clinical applications for viral, bacterial detection, and blood donor screening

What type of amplification are Transcription Mediated Amplification (TMA) and Nucleic Acid Sequence-Based Amplification (NASBA)

Both are isothermal RNA amplification methods.

How do TMA and NASBA differ?

They differ in the number of enzymes used.

What is the biological model for TMA and NASBA?

They are modeled after retroviral replication, utilizing reverse transcriptase (RT) enzyme.

Why is RNA used instead of DNA for amplification in TMA and NASBA?

• DNA: 2 copies per cell

• RNA: Many copies per cell (up to 10,000 copies/cell)

• Result: Amplification of RNA generates many cDNA copies via reverse transcription, increasing sensitivity.

What enzymes are used in TMA?

1. Reverse Transcriptase (RT) – generates ds cDNA from RNA and has RNase H activity

2. T7 RNA Polymerase – transcribes ds cDNA into many RNA copies

What is the role of reverse transcriptase in TMA?

Converts RNA target into cDNA and degrades RNA strand (RNase H activity), generating ds cDNA.

What is the role of T7 RNA polymerase in TMA?

A DNA-dependent RNA polymerase that recognizes the T7 promoter sequence and synthesizes many RNA copies from ds cDNA.

How is the amplification performed in TMA?

All steps occur at a single constant temperature (isothermal), producing exponential RNA amplification.

What is Nucleic Acid Sequence-Based Amplification?

A molecular biology method used to amplify RNA sequences.

What is a feature of conventional RT-PCR?

• Amplifies RNA using reverse transcriptase.

• Synthesizes a complementary DNA (cDNA) strand as a template.

• Requires a thermal cycler for reiterative (repetitive) PCR cycling.

Why is NASBA preferred over conventional RT-PCR in some applications?

It operates under isothermal conditions at a constant temperature of 41°C, eliminating the need for a thermal cycler.

What enzymes are used in NASBA?

1. Reverse Transcriptase

2. RNase H

3. T7 RNA Polymerase

What is the main advantage of NASBA in clinical diagnostics?

• Rapid diagnosis and high sensitivity.

• Works under constant temperature (41°C).

• Fewer manual steps, reducing contamination.

• Can be miniaturized for portable, battery-operated devices.

What are some clinical applications of NASBA?

• Quantification of HIV-1 in patient sera.

• Detection of bacteria such as:

  • Chlamydia trachomatis (common in young women).

  • Neisseria gonorrhoeae (causing STIs or STDs).

What are the advantages of NASBA over PCR?

• Faster and more sensitive in some cases.

• No need for temperature cycling.

• Reduced contamination risk due to fewer manual steps.

• Suitable for field or point-of-care testing using portable systems.

What are the two types of primers used in Strand Displacement Amplification (SDA)?

The two types are outer primers (bumper primers) and inner primers (SDA primers).

What are outer primers (bumper primers) in SDA, and where are they located?

They are located upstream of the SDA primers and are complementary to the target sequence.

What is the function of the outer (bumper) primers in SDA?

They displace the newly synthesized strand generated from the inner primer containing the restriction enzyme site, initiating strand displacement for amplification.

What is the function of the inner (SDA) primers?

They contain a recognition sequence for a nicking endonuclease and a sequence complementary to the target DNA.

What is the role of the restriction enzyme site in SDA?

The restriction enzyme site introduced by the inner primers allows nicking endonuclease activity, which creates single-strand breaks (nicks) needed to start synthesis of new strands.

How does exponential amplification occur in SDA?

As new strands are generated, they incorporate the restriction enzyme site, enabling continuous nicking and strand displacement — leading to exponential production of target copies.

What is Multiple Displacement Amplification (MDA)?

It is an isothermal DNA amplification method used to amplify the entire genome from very small or limited DNA samples using strand displacement activity of special DNA polymerases (e.g., Phi29 polymerase).

What are the clinical applications of MDA?

• Whole genome sequencing, allowing access to the entire genome from minimal samples.

• Single-cell genome sequencing, enabling genetic analysis from individual cells.

What is an example of MDA application?

• Single cells of uncultured bacteria

• Archaea

• Protists

• Individual viral particles

• Single fungal spores

What is the main purpose of the MDA technique?

The purpose of MDA is to overcome the problem of insufficient DNA in biological samples—particularly bacterial DNA in human biopsy specimens—making it possible to perform detailed molecular and genomic analyses.

How does the MDA reaction start?

By annealing random hexamer primers to different regions of the template genome.

Which enzyme carries out DNA synthesis in MDA?

Phage 29 DNA polymerase, a high-fidelity polymerase, carries out the DNA synthesis.

At what temperature is MDA performed?

MDA is isothermal, typically at 30°C or 40°C.

What are key features of Phage 29 DNA polymerase?

• High fidelity due to proofreading activity

• Strand displacement capability

• Allows small fragment generation from primers annealing at multiple genome sites

How does strand displacement and amplification occur in MDA?

• Primers anneal to the template and extend the DNA strand.

• Newly synthesized strands are displaced, exposing single-stranded regions.

• New random hexamers anneal to these displaced strands, and Phage 29 DNA polymerase synthesizes additional strands.

• This process of primer extension and displacement continues exponentially, amplifying the DNA.

Give an example of MDA application in clinical diagnostics.

MDA is used to amplify DNA from human colon and rectum biopsies for detection of Helicobacter pylori (H. pylori).

Why is MDA advantageous for detecting H. pylori?

H. pylori detection usually requires invasive techniques (endoscopy and biopsy), and MDA allows amplification from limited DNA in biopsy samples, enabling sensitive detection.

Do probe and signal amplification methods amplify the target DNA?

No, these methods do not amplify the target. Instead, either the probe or the signal is amplified.

What is probe amplification?

In probe amplification, the probe itself is amplified instead of the target sequence.

Give an example of a probe amplification method.

Ligase Chain Reaction (LCR)

How does LCR work?

It requires the probes to be ligated together using T4 DNA ligase to generate detectable signal.

What is signal amplification?

In signal amplification, the signal from the hybridized probe is amplified rather than the target sequence.

Give examples of signal amplification methods.

• Branched DNA (bDNA): Uses branched chain DNA probes to amplify the signal.

• Hybrid Capture: Uses anti-DNA-RNA hybrid antibodies to amplify detection signal.

What is the main advantage of probe and signal amplification methods?

They allow detection of nucleic acids without the need for target amplification, reducing risks associated with contamination and non-specific amplification.

How does LCR (Ligase Chain Reaction) work?

• Two DNA probes bind immediately adjacent to each other on the target template.

• The bound probes are ligated together using T4 DNA ligase.

• The ligated probes then serve as templates for additional probe binding and ligation, leading to amplification.

Why does LCR have greater specificity than conventional PCR?

Because T4 ligase only ligates probes that are perfectly complementary and adjacent, reducing non-specific amplification.

What are the clinical diagnostic applications of LCR?

• Chlamydia trachomatis

• Neisseria gonorrhoeae

• Sickle cell mutation (single base mutation detection)

Is LCR an isothermal amplification method?

No, LCR requires a thermal cycler, similar to conventional PCR.

What is the function of T4 DNA ligase in LCR?

T4 ligase facilitates the formation of phosphodiester bonds between:

• 5′ phosphate donor and 3′ hydroxyl acceptor

It allows:

• Sticky end joining

• Blunt end joining

What is the main purpose of LCR?

It is used for amplification of genomic DNA like PCR, but with greater specificity.

How is sickle cell mutation detected using LCR?

By using mutant-specific oligonucleotide primers that bind only to the mutation, ensuring highly specific detection.

What two key enzymes are required to drive the LCR reaction?

Both a DNA polymerase enzyme and a DNA ligase enzyme.

How many partial probes are used per DNA strand in LCR, and what happens to them?

Two partial probes are used per DNA strand and are ligated (joined) together to form a single probe.

Does signal amplification amplify the target DNA?

No, in signal amplification the target DNA is not enzymatically amplified. The detection sensitivity comes from amplification of the signal itself.

What are the main components of a bDNA signal amplification assay?

• bDNA amplifier probes – amplify the signal.

• bDNA target probes – bind specifically to the target nucleic acid.

• Capture probes – immobilize the target on a solid surface.

What technology is bDNA assay based upon?

Microtiter plate technology – it is a solid-phase signal amplification system.

How does the bDNA assay work?

• Capture probes are bound to the wells of the plate.

• Target-specific probes bind the nucleic acid of interest.

• Amplifier probes attach to the target probes and allow labeled molecules (e.g., alkaline phosphatase) to generate a detectable signal.

What are the clinical applications of bDNA assay?

• Detection of HIV nucleic acid

• Detection of Hepatitis C RNA

• Useful for detecting coinfections of HIV and Hepatitis C virus, which are common.

Why is bDNA considered a signal amplification method?

Because the target DNA is not amplified; instead, the signal generated from bound probes is amplified, increasing assay sensitivity.

What is the purpose of the Hybrid Capture Assay?

To detect DNA/RNA hybrid molecules using chemiluminescence detection with alkaline phosphatase (ALP) enzymes.

What are the two formats of Hybrid Capture Assay?

• Solution Hybridization Assay – does not require solid support.

• Antibody Capture Assay (Solid Capture) – requires a solid support such as microtiter wells or tubes.

What is the first step in the Hybrid Capture Assay?

Denature the DNA from the clinical specimen to make it single-stranded.

How is target DNA released from clinical specimens in Hybrid?

By treating specimens with a base solution (pH 8), which disrupts viruses or bacteria and releases the target DNA.

What happens after DNA denaturation?

The target DNA is hybridized with RNA probes, forming RNA:DNA hybrids.

How are RNA:DNA hybrids captured?

Capture antibodies coated on microtiter wells specifically bind to RNA:DNA hybrids, immobilizing them.

How is the signal detected in Hybrid Capture Assay?

Captured hybrids are detected with antibodies conjugated to alkaline phosphatase (ALP).

• ALP catalyzes a chemiluminescent substrate, producing a measurable signal.

Give a clinical example of Hybrid Capture Assay application.

Detection of Human Papillomavirus (HPV), a DNA virus causing sexually transmitted infections (STI).

Why is chemiluminescence used in HCA?

It amplifies the detection signal, allowing sensitive measurement of RNA:DNA hybrids without amplifying the target nucleic acid.