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MIC661:MOLECULAR DIAGNOSTIC > C3 > Flashcards

C3 Flashcards

1
Q

Components & its functions in PCR

A
  • 2 primer: direct DNA synthesis
  • dNTPs: building block to extend primer
  • KCl: optimal hybridisation of primer to template
  • Buffer: maintain optimal pH for enzyme reaction
  • DNA polymerase: extend primer (add dNTP)
  • Template DNA: sample DNA being tested
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2
Q

Principle of PCR

A
  • Primer are design to be complementary to end of GOI
  • Result in exponential increase in total number of target DNA copy
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3
Q

Procedure of PCR

A

Denature
- DNA template heated to 96C
- Break weak hyrogen bond
- Result in ssDNA

Annealing
- Mixture cooled to 50-70C
- Allow primer to bind with their complementary sequence in template

Extension
- Heated to 68-72C
- Optimal tempt for DNA polymerase
- Extend the primer by adding dNTPs in sequential manner

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4
Q

Tips & points to ponder on PCR concepts

A
  • Primer design
  • GC content: primer should be 45-55% GC
  • Melting temp: Both primer must have similar temp
  • PCR contamination & troubleshooting
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5
Q

Pros & cons of PCR

A

Pros
- Extremely sensitive
- Number of variations of PCR method can be expand the utility of this methodology

Cons
- Potential contamination
- Eg: high DNA conc, DNA template control/ standard

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6
Q

Application of PCR

A

Molecular identification
- DNA fingerprinting
- Genotyping
- Drug discovery

Sequencing
- Bioinformatics
- Human genome project

Genetic Engineering
- Gene expression studies

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7
Q

Variants of PCR

A
  • PCR modifications
  • RT-PCR
  • Real Time PCR
  • Nested PCR
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8
Q

Explain multiplex PCR

A
  • Amplify multiple target sequences in single PCR reaction using multiple primer sets
  • Time saving & less effort to obtain result
  • Poor sensitivity & specificity
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9
Q

Application of multiplex PCR

A
  • Detect deletions, polymorphism & mutation
  • Detect different virus, bacteria & pathogens
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10
Q

Comparison between singleplex & multiplex PCR

A

Singleplex
- Simple & fast
- Single target
- Costly
- Time consuming

Multiplex
- Complex- multiple primer needed
- Multiple target
- Cheap
- Time saving

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11
Q

Explain sequence specific PCR

A
  • Involve design one or both primer so that they will or will not allow amplification (3 mismatched principal)
  • Use to match patient & donor for bone marrow & cord blood transplant
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12
Q

Explain RT PCR

A
  • RNA template is reverse transcribe to generate cDNA using retroviral reverse transcriptase
  • cDNA amplified by PCR
  • Detection using fluorescent dye or agarose gel electrophoresis (ethidium bromide stain)
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13
Q

2 basic reaction involved in RT PCR are

A
  • cDNA is synthesised from mRNA template by AMV or MMLV reverse transcriptase
  • Second cDNA strain is synthesised & subsequent PCR amplification is performed with Taq DNA polymerase
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14
Q

Explain one step & two step RT PCR

A

One step
- Combine first strand cDNA synthesis (RT) & subsequent PCR in single reaction

Two step
- Two separate reactions
- Begin with first starnd cDNA synthesis (RT)
- Followed by cDNA amplification by PCR

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15
Q

Comparison of one step & two step RT PCR

A

One step
- Primer: Gene specific
- Analysis of 1/2 gene
- Fast, convenient

Two step
- Primer: Oligo (dT), hexamers, gene specific
- Analysis of multiple genes
- Flexible

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16
Q

Pros & cons of RT PCR

A

Pros
- Sensitivity
- Quantitation
- Sample integrity requirements

Cons
- Sample purity requirements: no contam
- Optimisation is difficult

17
Q

Application of RT PCR

A
  • Use to create cDNA libraries
  • Most sensitive techniques for mRNA detection & quantitation
18
Q

Explain Real time PCR

A
  • Based on detection & quantification of fluorescent signal
  • Use fluorescent signal by special probes or DNA binding dyes
19
Q

How to measure the result of Real Time PCR

A
  • Fluorescent signal created indirect proportion to amount of PCR product formed
  • Measured repeatedly with each PCR cycle
  • Measurement performed during amplification process
20
Q

Which PCR able to amplify & detect changes in amplicon concentration

A
  • Real Time PCR
  • Quantitative PCR
  • qPCR
21
Q

What is fixed fluorescent threshold in Real Time PCR

A

Set significantly above the baseline fluorescent level & can be alter by operator

22
Q

What is parameter CT (threshold cycle)

A

Cycle number at which fluorescent emissions exceed the fixed threshold

23
Q

Pros of Real Time PCR quantitation

A
  • Eliminates post PCR processing of PCR product
  • Help increase throughput
  • Reduce chances of carry over contamination
  • Can be achieved over a large range of initial target concentrations
24
Q

Explain curve in Real time PCR

A
  • Distinguish amplified product through plotting fluorescent as function of temperature
  • Each curve represents different amplified product
25
Q

Types of detection use in Real Time PCR

A
  • SYBRGreen
  • TaqMan
26
Q

Comparison SYBRGreen between TaqMan

A

SYBRGreen
- Require no probe
- Cheap
- Readily available

TaqMan
- More expensive
- Can detect different report
- Reduce chance of non specific & contamination products

27
Q

Application of Real Time PCR

A
  • Quantitation of gene expression
  • Detection of X chromosomes
  • Haplotyping
  • Quantitative microsatellite analysis
28
Q

Pros & cons of Real time PCR

A

Pros
- High sensitivity
- High degree of reproducibility
- Multiplexing capability

Cons
- Require special instrument
- Increase risk of false negative value (no internal std)
- Susceptible to PCR inhibition

29
Q

Explain Nested PCR

A
  • Modification of PCR
  • 2 internal primer use to amplify PCR products of 2 external primers
  • Increase fold of amplification & specificity
30
Q

Procedure of nested PCR

A
  • 2 sets of primer & 2 successive PCR procedures
  • Second primer set function to amplify secondary target within the first run product
  • Target DNA undergo first round of PCR with first set of primer
  • Amplicon from first reaction undergo second round amplification with the second set of primer
31
Q

Types of nested PCR

A
  • Fully nested
  • Semi nested: reuse one primer from first round in second round & increase sensitivity & specificity
32
Q

Application of nested PCR

A
  • Detection of microorganism when they are present in very low quantities
  • Commercial applications
  • Nested with multiplex- 2 round containing multiple primer for different target
33
Q

Pros & cons of nested PCR

A

Pros
- Increase sensitivity
- Increased specificity: enrichment of target seq in first round

Cons
- Highly susceptible to contamination
- Cannot be used for quantitative analysis of target

34
Q

Difference between PCR & Nested PCR

A

PCR
- Less sensitive
- Cheap
- Time saving
- Non specific

Nested PCR
- More sensitive
- Expensive
- Time consuming
- Specific

MIC661:MOLECULAR DIAGNOSTIC (7 decks)

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