Real-time PCR Techniques

Real-time PCR

• Real-time PCR (qPCR) monitors PCR reactions in real time, allowing for quantification of the DNA, cDNA or RNA product.
• It's a technique used to detect the progress of a PCR reaction in real time. Small amounts of PCR product can be quantified simultaneously.
• Real-time PCR uses fluorescence to detect the amplification of the target DNA. A reporter molecule produces fluorescence as the reaction progresses. The increase in fluorescence is directly proportional to the accumulation of the PCR product.

Lecture Outline

• What is real-time PCR?
• Nucleic acid sequence-based amplification
• Branched DNA detection

Automated PCR and Detection

• The COBAS Amplicor Analyzer automates the process of PCR amplification and product detection.
• It's a benchtop system that fully automates amplification and detection using polymerase chain reaction (PCR) testing.
• The system combines a thermal cycler, automatic pipettor, incubator, washer, and reader.
• It has a 48 sample capacity per run, supports multiplexing tests (up to 6 detections per sample), and uses an AmpErase enzyme for selective amplification by eliminating uracil.
• It's used in infectious disease applications (for HIV, HCV, HBV, CMV, Chlamydia, Neisseria, and Mycobacterium tuberculosis).

Real-time or Quantitative PCR (qPCR)

• qPCR is a technique to monitor the progress of a PCR reaction and quantify a small amount of DNA, cDNA or RNA in real time.
• The fluorescence produced by a reporter molecule increases as the reaction proceeds.
• The amount of the PCR product increases in each cycle of amplification.

Real-Time or Quantitative PCR (qPCR) - Additional Points

• Standard PCR can be modified to be real-time by adding a probe or dye to generate a fluorescent signal from the product.
• Real-time detection allows for precise quantification of starting material.
• It is performed in specialized thermal cyclers with fluorescent detection systems.

Quantitative PCR (qPCR) - Detailed Analysis

• The PCR product increases exponentially.
• The qPCR signal is shown as an exponential curve with phases like; a lag phase, a linear phase, and a stationary phase.
• The length of the lag phase is inversely proportional to the amount of starting material.

Sequence Detection Applications

• End-point PCR: Simple, produces results like positive (+) or negative (-) for a variety of applications. Detection of PCR products including pathogens and transgenes, genotyping (allelic discrimination) of Single Nucleotide Polymorphisms (SNPs).
• Real-time PCR: Comprehensive results that include quantified absolute and relative measurements. Quantitative PCR can also be used for optimization and hybridization analysis to evaluate probe hybridization.

qPCR Detection Systems

• Specific dyes and probes are used to detect and measure the amplification of the intended DNA product.
  • DNA-specific dyes (e.g., ethidium bromide, SYBR Green) fluoresce when bound to double-stranded DNA, but this isn't very specific.
  • Hybridization probes, are designed to target a specific sequence of DNA and only bind and fluoresce the intended PCR product.
  • Primer-incorporated probes bind to the PCR product and label it.

qPCR Detection Systems (continued)

• Cleavage-based probes: TaqMan assay uses a fluorescent reporter and a quencher molecule.
• Molecular beacons: Closed hairpin loop probe structure with fluorescence reporter at one end and a quencher molecule at the other end.
• FRET probes (Fluorescence Resonance Energy Transfer): Two fluorescent dyes are used, where energy is transferred between them in the presence of the target sequence. This increases the fluorescence of one dye.

Additional Information

• Several types of detection systems are possible for qPCR analysis, including thermal cyclers with specialized software and the use of plates, tubes, and capillaries for reactions.
• The use of the COBAS Amplicor Analyzer, the LightCycler, the iCycler, and the 7700 Applied Biosystems are different qPCR instruments.

PCR Advantages

• The technique is generally specific, simple, rapid and inexpensive.
• It can amplify from low quantities of DNA.
• It works with damaged DNA.
• The technique is sensitive and flexible.

PCR Limitations

• Contamination risk,
• Primer complexities,
• Primer-binding site complexities,
• Amplifies rare but important species, or
• Detection method complexities.

This quiz explores the fundamentals of real-time PCR, including its mechanisms for quantifying DNA, cDNA, and RNA. Learn about the role of fluorescence in monitoring PCR reactions and the automation of the process using systems like the COBAS Amplicor Analyzer.