Define amplification
Process which nucleic acid molecules is enzymatically copied to generate progeny with the same sequence as their parent
Explain diagnosis based on DNA amplification methods
- Molecular diagnostic assay require amplification of nucleic acid, signal amplification or both
- Eg tools: PCR, SDA & transcription mediated
Explain amplification methods
- Rapid, specific & very sensitive
- Significant precautions must taken to avoid contamination
Type of diagnosis based on DNA amplification methods
- Signal amplification
- Target amplification
Define signal & target amplification
Signal amplification
- Probe based methods that employ enhancement of a target specific signal w/o raising target copy number
Target amplification
- Detect & amplify gene of interest
- Target amplify over & over & final signal dependent on amplified target
Pros & cons single amplifications
Pros
- Can be quantify
- Reproducible
- No sample extraction & purification
Cons
- Time consuming
- Less sensitive
- Limited dynamic range
Pros & cons of target amplification
Pros
- Broad dynamic range
- More sensitive
- Time saving
Cons
- Poor reproducibility
- Cant be quantified
- Require sample extraction & purification
Types of signal amplification methods
- Branched DNA (bDNA)
- Rolling circle amplification (RCA)
- Ramifications amplification (RAM)
- LAMP
- Invader Cleavase Technology
Target of bDNA
Entire genome but amount of DNA & RNA did not increase
Application bDNA
- To detect infectious agent in clinical material
- Detect & measure expression of cellular mRNA
Principle of bDNA
- Use dozen of probes that helps the attachments of signal amplification molecule to viral nucleic acid target
- Bind to specific target probe & linked to alkaline phosphate (ALP)
- ALP catalyst chemiluminescence reaction
Explain Procedure in bDNA
- Target DNA & RNA isolated. dsDNA denature into ssDNA. RNA or ssDNA capture to a microwell by first set of target probes which bind to capture probes coated on microwell
- Second set of target probes hybridise to DNA /RNA
- Second set of target probes hybridise to branched DNA. Multiple copy of ALP labeled probe hybridise to branched DNA molecule
- Detection is achieved by incubating the ALP bound complex with chemiluminescent substrate
Procedure of bDNA
- Sample preparation
- Target hybridisation
- Signal amplification
- Detection
Pros & cons of bDNA
Pros
- Very specific
- Quantitative
- Original amount of target remain unchanged
Cons
- Expensive
- Difficult procedure
- Low sensitivity compare to target & signal amplification
Explain rolling circle amplification (RCA)
- Simple & isothermal amplification techniques
- Amplification mode is linear & one primer is use
- Based on in vivo rolling circle replication of bacteriophage
RCA can generate
Thousand of repeating DNA sequences using circular templates under catalysts of DNA polymerase
Procedure of RCA
- Once C probes hybridise to 5-3 end are placed, a closed circular molecules generate following the incubation of C-probe target complex w DNA ligase
- Resulting closed circular molecule is helically twisted around the target strand
- Unique design of C probe allow amplification by RCA where a single forward primer complementary to linker region of C probe & DNA polymerase bearing strand displacement are employed
Why RCA considered a signal amplification method
- Amplification of signal: generate many copies of target DNA sequence, easy to detect
- Enhanced sensitivity: can detect low conc of target DNA
- Specificity: rely on binding of primer to specific region of circular DNA template
- Versatility
DNA quantification in RCA
- Design as a reference standard
- Amplification of both target & reference
- Signal detection
- Comparison
Application of RCA
- Use to detect protein
- Use in in situ detection of mRNA
- Combination of RCA & DNA microarray allow real time detection of multiple targets with great sensitivity & specificity
Pros & cons of RCA
Pros
- Ideal method for in situ amplified (not need thermal cycling)
- Can make target capture, amplification reaction & detection on same solid support
- Reaction under isothermal conditions
Cons
- Sensitivity is lower than RAM
- Difficult to achieve quantitative detection
- Difficult procedure
What is RAM
Novel isothermal DNA amplification method that amplifies a C probe exponentially through the mechanism of primer extension, strand displacement & ramifications
RAM is also known as
- Hyperbranched rolling circle amplification
- Cascade rolling circle amplification
Procedure of RAM
- Open loop covalently linked by T4 DNA ligase in target dependent manner producing closed DNA circle
- Circular DNA template for forward primer to attach. DNA polymerase extends the bound forward primer with C probe & displace downstream DNA generating multimeric ssDNA
- Multimeric ssDNA serve as template for reverse primer to hybridise, extend & disolace downstream DNA generating large ramified DNA complex
- Ramifications process end when all ssDNA becomes dsDNA & no new primer available
