Overview of Real-Time PCR

Overview of Real-Time PCR

• Real-time PCR is a variation of the standard Polymerase Chain Reaction (PCR) technique.

• Commonly used to quantify DNA or RNA in a sample.

  • Employs sequence-specific primers for determination of copy number.

• Quantification is achieved by measuring the amount of amplified product at each stage during the PCR cycle.

Amplification Dynamics

• If a particular DNA or RNA sequence is abundant in the sample, amplification is observed in earlier cycles.

• If the sequence is scarce, amplification is observed in later cycles.

• Quantification of the amplified product is obtained using:

  • Fluorescent probes

  • Fluorescent DNA-binding dyes

Components of Real-Time PCR

Key Components:

• Primers

• Deoxynucleotide triphosphates (dNTPs)

• Reverse transcriptase

• DNA polymerase

• Water and buffer

• RNA Template

• RT + PCR

• Amplified cDNA

Step One: Reverse Transcription

• Total RNA is reverse transcribed into complementary DNA (cDNA).

• The enzyme utilized is generally reverse transcriptase (RT).

• This step maintains the same amount of total RNA present.

• There are different priming strategies for first-strand cDNA synthesis:

  • Random primers

  • Oligo(dT) primers

  • Gene-specific primers (GSPs)

• Random primers or a combination of oligo(dT) and random primers are most commonly used.

Importance of Reverse Transcriptase

• Reverse transcriptase is critical for the success of quantitative reverse transcription PCR (qRT-PCR) as is DNA polymerase.

• Selection criteria for RT:

  • High yields of full-length cDNA.

  • Good activity at high temperatures essential for RNA denaturation with secondary structure.

• In one-step qRT-PCR, RT must still be active at higher temperatures to:

  • Use GSPs with high melting temperatures (Tm), increasing specificity and reducing background noise.

Overview of Real-Time PCR Components

DNA Polymerase

• The performance of PCR is dependent on using a thermostable DNA polymerase.

• Taq DNA Polymerase is notable for having residual activity at low temperatures.

• The potential for nonspecific primer annealing to DNA during reaction setup can lead to synthesis of nonspecific products.

• Mis-priming issues can be minimized by the use of a “hot-start” enzyme:

  • Ensures DNA polymerase is inactive during the setup and initial DNA denaturation steps.

• DNA polymerase operates on the template DNA in the 3’-5’ direction and synthesizes a complementary strand in the 5’-3’ direction.

Deoxynucleotide Triphosphates (dNTPs)

• dNTPs are essential raw materials for synthesizing new DNA products.

• They are utilized by Taq polymerase in the synthesis process.

Magnesium

• Magnesium ions act as a co-factor for thermostable DNA polymerase and are generally used at a final concentration of:

  • 3 mM

• Presence of proteins and chelating agents in samples can reduce magnesium concentration, inhibiting PCR reactions.

Template DNA

• Recommended usage of template nucleic acid per real-time PCR reaction:

  • Between 10 and 1,000 copies.

  • Equivalent to approximately 100 picograms (pg) to 1 microgram (μg) of genomic DNA.

  • cDNA generated from 1 pg to 100 ng of total RNA.

• An excess of template may increase contamination levels, affecting PCR efficiency negatively.

• RNA templates should be treated to reduce genomic DNA contamination, typically through DNase treatment.

Real-Time Fluorescent PCR Chemistries

TaqMan® Reaction Components

• TaqMan reaction includes:

  • A pair of unlabeled primers.

  • A TaqMan probe labeled with:

  • FAM™ or VIC™ dye at the 5' end

  • A minor groove binder (MGB)

  • A nonfluorescent quencher (NFQ) at the 3' end

  • All other components necessary for PCR reactions.

TaqMan® Reaction Process

1. The temperature is raised to denature double-stranded cDNA at the start of real-time PCR. During this step, the fluorescent dye on the 5' end of the TaqMan probe is quenched by the NFQ on the 3' end.

2. The reaction temperature is then lowered for primers and probes to anneal to their target sequences.

3. Taq DNA polymerase synthesizes new strands using unlabeled primers and the template. When it encounters a TaqMan probe, its endogenous 5' nuclease activity cleaves the probe:

   • This cleavage separates the fluorescent dye from the quencher, leading to a measurable increase in signal intensity.

Signal and Fluorescence Measurement

• Graphs can reflect relation of signal intensity against cycle number to demonstrate amplification performance.

• Example signal intensity values given are:

  • Cycle 1: 4,000

  • Cycle 2: 3,500…

SYBR® Green Dye in Real-Time PCR

Characteristics of SYBR® Green

• SYBR Green I dye binds to the minor groove of double-stranded DNA (dsDNA).

• When bound, the dye exhibits a 1,000-fold greater fluorescence than when unbound.

• Fluorescence increases proportionally as more amplicons accumulate during PCR.

• Requires highly specific primers for accurate qPCR results.

• Nonspecific product formation can lead to false positives due to the broad detection nature of SYBR Green signal.

Workflow Stages with SYBR® Green Dye

1. Denaturation

2. Annealing

3. Extension

4. Fluorescence Tagging

Specific Examples and Data

• Example of amplification plots with numerical thresholds and coefficients is illustrated, describing:

  • Slope: -3.364

  • Y-intercept: 42.90

  • Correlation coefficient: 0.999

• Starting quantity and background thresholds are visually represented on amplification plots across various cycle numbers.

Conclusion

• Understanding real-time PCR involves comprehending various components, their functions, and interactions during the amplification cycle. Accurate quantification hinges on the careful selection of enzymes, primers, and cycling conditions.