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MIC661:MOLECULAR DIAGNOSTIC > C2 > Flashcards

C2 Flashcards

1
Q

Define amplification

A

Process which nucleic acid molecules is enzymatically copied to generate progeny with the same sequence as their parent

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2
Q

Explain diagnosis based on DNA amplification methods

A
  • Molecular diagnostic assay require amplification of nucleic acid, signal amplification or both
  • Eg tools: PCR, SDA & transcription mediated
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3
Q

Explain amplification methods

A
  • Rapid, specific & very sensitive
  • Significant precautions must taken to avoid contamination
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4
Q

Type of diagnosis based on DNA amplification methods

A
  • Signal amplification
  • Target amplification
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5
Q

Define signal & target amplification

A

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

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6
Q

Pros & cons single amplifications

A

Pros
- Can be quantify
- Reproducible
- No sample extraction & purification

Cons
- Time consuming
- Less sensitive
- Limited dynamic range

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7
Q

Pros & cons of target amplification

A

Pros
- Broad dynamic range
- More sensitive
- Time saving

Cons
- Poor reproducibility
- Cant be quantified
- Require sample extraction & purification

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8
Q

Types of signal amplification methods

A
  • Branched DNA (bDNA)
  • Rolling circle amplification (RCA)
  • Ramifications amplification (RAM)
  • LAMP
  • Invader Cleavase Technology
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9
Q

Target of bDNA

A

Entire genome but amount of DNA & RNA did not increase

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10
Q

Application bDNA

A
  • To detect infectious agent in clinical material
  • Detect & measure expression of cellular mRNA
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11
Q

Principle of bDNA

A
  • 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
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12
Q

Explain Procedure in bDNA

A
  • 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
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13
Q

Procedure of bDNA

A
  • Sample preparation
  • Target hybridisation
  • Signal amplification
  • Detection
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14
Q

Pros & cons of bDNA

A

Pros
- Very specific
- Quantitative
- Original amount of target remain unchanged

Cons
- Expensive
- Difficult procedure
- Low sensitivity compare to target & signal amplification

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15
Q

Explain rolling circle amplification (RCA)

A
  • Simple & isothermal amplification techniques
  • Amplification mode is linear & one primer is use
  • Based on in vivo rolling circle replication of bacteriophage
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16
Q

RCA can generate

A

Thousand of repeating DNA sequences using circular templates under catalysts of DNA polymerase

17
Q

Procedure of RCA

A
  • 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
18
Q

Why RCA considered a signal amplification method

A
  • 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
19
Q

DNA quantification in RCA

A
  • Design as a reference standard
  • Amplification of both target & reference
  • Signal detection
  • Comparison
20
Q

Application of RCA

A
  • 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
21
Q

Pros & cons of RCA

A

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

22
Q

What is RAM

A

Novel isothermal DNA amplification method that amplifies a C probe exponentially through the mechanism of primer extension, strand displacement & ramifications

23
Q

RAM is also known as

A
  • Hyperbranched rolling circle amplification
  • Cascade rolling circle amplification
24
Q

Procedure of RAM

A
  • 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
25
Q

Application of RAM

A
  • Detect target nucleic acid in clinical sample (C.trachomatis)
  • Use to identify E.coli in human & food sample
26
Q

Pros & cons of RAM

A

Pros
- Ideal for in situ amplification (no thermal cycling)
- Can make target capture, amplification reactions & detection happen in same solid support
- Million fold amplification feasible in short time

Cons
- Low specificity
- Difficult procedure
- Nonspecific background signal

MIC661:MOLECULAR DIAGNOSTIC (7 decks)

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