Real-time PCR & Molecular Diagnostics Flashcards
How is PCR used in virology?
In vitro nucleic acid amplification tests are available for the quantification of Hep C virus (HCV) RNA in human serum or plasma. -this test is intended for use in conjunction with clinical presentation and other laboratory markers to assess patient viral response to antiviral treatment as measured by changes in serum or plasma HCV RNA levels.
Also Chlamydia trachomatis and Neisseria gonorrhoea can be detected using PCR.
There is also an in vitro nucleic acid amplification test for the quantification of Hep B virus (HBV) DNA in human serum or plasma.
What is the formula for PCR?
N=N0 x 2n
N= number of amplified molecules N0= initial number of molecules n= number of amplification cycles
What are the features for real-time PCR?
Quantitative
Sensitive
Specific
Reproducible quantitation of nucleic acids.
Give an example of one detection format.
The lightCycler 2.0 system. Uses real-time PCR to quantify the starting target material, i.e, it correlates the increase in fluorescent signal, measured at each amplification cycle, to the amount of PCR product formed.
Give two ways in which PCR products can be detected fluorescently.
1) . Intercalating fluorescent dye, SYBR green 1.
2) . Fluorescently labelled, target-specific probes.
List the advantages and disadvantages for using SYBR green I format.
Advantages: economical, easy to use, sensitive
Disadvantages: binds to any double stranded DNA molecules (risk of overestimation).
What are the three good aspects of using the LightCycler?
Rapid: assay and analysis are complete in less than 45 minutes.
Accurate: ultra-precise hardware and optimised software to give accurate and highly reproducible results.
Sensitive: expect a greater signal-to-noise ratio and fewer unwanted PCR products.
What are the advantages of using real-time multiplex PCR for diagnosis?
Can analyse multiple genes simultaneously within a single reaction.
Provide internal controls
Lower reagent costs
Preservation of precious samples
What is PCR?
Allows massive replication of a target sequence of DNA and allows detection of DNA or RNA sequences that are present as only one or two copies per cell.
What are the steps for performing PCR?
The target sequence of DNA is extracted from the cell and denatured into its single strand by heating to 90’C. Two oligonucleotide primers complementary to the opposing ends of the single strands of the target DNA are synthesised and annealed. Primers for PCR consist of a 20-30 nucleotide sequence specific for the target DNA and result in the amplification of that sequence of DNA only. The primed single strand is then extended using DNA polymerase 1. The newly synthesised double-stranded sequence is then denatured by heating to 90’C and new oligonucleotide primers added. This process is repeated through 25 to 30 cycles and results in a large increase in DNA.
What is a thermocycler?
Allows the automatic control of the rate of heating and cooling, the incubation times at each temperature and the number of cycles.
How is the PCR product detected?
Using gel electrophoresis and autoradiography.
What is RT-PCR?
mRNA is extracted from the cells or tissue, converted to cDNA using the enzyme reverse transcriptase and PCR is then carried out. This method is used to detect the expression of specific mRNA sequences in cells or tissues.
How is SYBR green I used in PCR?
During annealing, PCR primers hybridise to the target DNA sequence and form small regions of dsDNA where SYBR green I intercalates (mixing in) the fluorescent signal.
In the elongation phase, more dsDNA is formed and more SYBR green I dye can intercalate. Producing a higher fluorescent signal.
At the end of the elongation phase, all DNA has become double stranded and the maximum amount of SYBR green I is intercalated. The fluorescence is measured at the end of each elongation phase.
What are the application fields for RT-PCR?
Gene detection and expression for S. aureus.
Genotyping and gene knockdown (experimental technique by which the expression of one or more of an organisms genes are reduced) for Zea Mays (plant).
