C3 Flashcards
Components & its functions in PCR
- 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
Principle of PCR
- Primer are design to be complementary to end of GOI
- Result in exponential increase in total number of target DNA copy
Procedure of PCR
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
Tips & points to ponder on PCR concepts
- Primer design
- GC content: primer should be 45-55% GC
- Melting temp: Both primer must have similar temp
- PCR contamination & troubleshooting
Pros & cons of PCR
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
Application of PCR
Molecular identification
- DNA fingerprinting
- Genotyping
- Drug discovery
Sequencing
- Bioinformatics
- Human genome project
Genetic Engineering
- Gene expression studies
Variants of PCR
- PCR modifications
- RT-PCR
- Real Time PCR
- Nested PCR
Explain multiplex PCR
- Amplify multiple target sequences in single PCR reaction using multiple primer sets
- Time saving & less effort to obtain result
- Poor sensitivity & specificity
Application of multiplex PCR
- Detect deletions, polymorphism & mutation
- Detect different virus, bacteria & pathogens
Comparison between singleplex & multiplex PCR
Singleplex
- Simple & fast
- Single target
- Costly
- Time consuming
Multiplex
- Complex- multiple primer needed
- Multiple target
- Cheap
- Time saving
Explain sequence specific PCR
- 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
Explain RT PCR
- 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)
2 basic reaction involved in RT PCR are
- 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
Explain one step & two step RT PCR
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
Comparison of one step & two step RT PCR
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
Pros & cons of RT PCR
Pros
- Sensitivity
- Quantitation
- Sample integrity requirements
Cons
- Sample purity requirements: no contam
- Optimisation is difficult
Application of RT PCR
- Use to create cDNA libraries
- Most sensitive techniques for mRNA detection & quantitation
Explain Real time PCR
- Based on detection & quantification of fluorescent signal
- Use fluorescent signal by special probes or DNA binding dyes
How to measure the result of Real Time PCR
- Fluorescent signal created indirect proportion to amount of PCR product formed
- Measured repeatedly with each PCR cycle
- Measurement performed during amplification process
Which PCR able to amplify & detect changes in amplicon concentration
- Real Time PCR
- Quantitative PCR
- qPCR
What is fixed fluorescent threshold in Real Time PCR
Set significantly above the baseline fluorescent level & can be alter by operator
What is parameter CT (threshold cycle)
Cycle number at which fluorescent emissions exceed the fixed threshold
Pros of Real Time PCR quantitation
- 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
Explain curve in Real time PCR
- Distinguish amplified product through plotting fluorescent as function of temperature
- Each curve represents different amplified product
Types of detection use in Real Time PCR
- SYBRGreen
- TaqMan
Comparison SYBRGreen between TaqMan
SYBRGreen
- Require no probe
- Cheap
- Readily available
TaqMan
- More expensive
- Can detect different report
- Reduce chance of non specific & contamination products
Application of Real Time PCR
- Quantitation of gene expression
- Detection of X chromosomes
- Haplotyping
- Quantitative microsatellite analysis
Pros & cons of Real time PCR
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
Explain Nested PCR
- Modification of PCR
- 2 internal primer use to amplify PCR products of 2 external primers
- Increase fold of amplification & specificity
Procedure of nested PCR
- 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
Types of nested PCR
- Fully nested
- Semi nested: reuse one primer from first round in second round & increase sensitivity & specificity
Application of nested PCR
- Detection of microorganism when they are present in very low quantities
- Commercial applications
- Nested with multiplex- 2 round containing multiple primer for different target
Pros & cons of nested PCR
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
Difference between PCR & Nested PCR
PCR
- Less sensitive
- Cheap
- Time saving
- Non specific
Nested PCR
- More sensitive
- Expensive
- Time consuming
- Specific
