Activity 4-3 - Real-Time Quantitative Polymerase Chain Reaction
- Page ID
- 158621
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Distinguish between qPCR and RT-PCR, and understand how they are connected.
- Explain how SYBR Green is used to detect and quantify DNA amplification.
- Describe the thermal cycling steps in a qPCR reaction.
- Set up a qPCR reaction using primers, cDNA, and master mix.
- Interpret Ct values and melting curves to assess gene expression and amplification quality.
qPCR (Quantitative PCR): A method to detect and measure the amount of a specific DNA sequence in real time using fluorescence.
RT-PCR (Reverse Transcription PCR): A method that converts RNA into cDNA, often used before qPCR to study gene expression.
SYBR Green: A fluorescent dye that binds to double-stranded DNA, used to monitor amplification during qPCR.
Ct Value (Cycle Threshold): The cycle number at which the fluorescence signal exceeds background; inversely related to the amount of target DNA.
Melting Curve Analysis: A post-PCR step that slowly heats the sample to identify the specificity of the amplified product.
Primer-Dimers: Unintended short double-stranded DNA products formed by primers binding to each other instead of the target sequence.
- What is the main difference between RT-PCR and qPCR?
- Why is it useful to measure gene expression instead of just checking if a gene is present?
- What might happen if primers are not designed specifically for your gene of interest?
- What does a lower Ct value tell you about gene expression?
Real-Time Quantitative Polymerase Chain Reaction (qPCR)
Quantitative Polymerase Chain Reaction (qPCR), sometimes called Real-time PCR, should not be confused with Reverse Transcription PCR (RT-PCR). Once the cDNA has been synthesized from RT-PCR, we can perform quantitative PCR (qPCR) to determine how much of a specific gene is present. Unlike regular PCR, which only shows if a gene is present or absent, qPCR gives us real-time, quantitative information about gene expression. This is done using a special dye called SYBR Green, which binds to double-stranded DNA. As PCR amplifies the cDNA, more double-stranded DNA is created, and the fluorescence from SYBR Green increases. A qPCR machine detects this fluorescence during each cycle, giving a continuous readout of DNA amplification. Let’s return to our Bt corn example. If we run qPCR with primers specific to the Bt gene and see a strong fluorescent signal, it tells us the gene is not just present — it is being highly expressed in the tissue we tested. This gives us quantitative evidence of gene activity.
The thermal cycling conditions for qPCR are carefully controlled. Each cycle includes a denaturation step at 95°C, where double-stranded DNA separates into single strands; an annealing step at 55°C, where primers bind to their target sequences; and an extension step at 72°C, where the DNA polymerase builds new strands. These steps are repeated for about 40 cycles. After the amplification is complete, a melting curve analysis is performed. During this step, the sample is slowly heated, and the qPCR machine tracks the drop in SYBR Green fluorescence as the DNA melts (denatures). A sharp, single melting peak indicates a specific product, while multiple peaks or irregular curves may suggest nonspecific amplification or primer-dimers.
Image of a flow chart summarizing Real-time quantitative PCR. Image created by Dr. Victor Pham's student, Diana Valdovinos.
qPCR Protocol with SYBR Green
Objective: Use qPCR to detect and measure how much of a target gene is being expressed in a genetically modified sample by using cDNA and SYBR Green dye.
Materials:
- SYBR Green Master Mix (2X)
- Forward primer (200 µM stock)
- Reverse primer (200 µM stock)
- Synthesized cDNA sample (from RT step)
- Nuclease-free water
- qPCR tubes or 96-well plate
- qPCR machine
Procedure:
- In a clean tube, create a qPCR MasterMix-Primer name with your group by mixing the following solution: (Note: You need to create another qPCR MasterMix for different primers)
- 10 µL of SYBR Green Master Mix (1X final concentration)
- 5 µL of your primer mix (from Activity 3.1, or 20 µM Forward and 20uM Reverse Primers)
- Add 15 µL of the qPCR Master Mix to your qPCR tube (Note: qPCR tube contains a flat cap)
- Into your qPCR tube:
- Add up to 5 µL of cDNA (50 ng)
- Adjust the remaining volume to 5uL with Nuclease-free water
- Add up to 5 µL of cDNA (50 ng)
- Load the tubes/plate into the qPCR machine and run this program:
| Step | Temp (°C) | Time | Purpose |
|---|---|---|---|
| Initial Denaturation | 95°C | 10 sec | Unwind DNA strands |
| Annealing | 55°C | 30 sec | Primers bind to DNA |
| Extension | 72°C | 30 sec | DNA is copied |
| Repeat above steps for 40 cycles | |||
| Increase the temperature slowly from 55°C to 95°C | Go up 0.5°C every 5 seconds | ||
| Final SYBR Imaging |
- The qPCR machine will detect fluorescence from SYBR Green, which binds to double-stranded DNA.
- The more gene expression, the earlier the signal appears (lower Ct value).
- Use the software to create a bar graph of expression levels across samples.
- You prepared a qPCR reaction using a cDNA template and primers specific to your gene of interest.
- You ran thermal cycling conditions and monitored fluorescence using SYBR Green.
- You analyzed Ct values and created a bar graph to visualize expression across samples.
- You checked the specificity of your results using melting curve analysis.
- qPCR allows for quantitative measurement of gene expression.
- SYBR Green fluorescence is directly tied to double-stranded DNA production—more fluorescence = more gene product.
- A sharp, single melting peak indicates a specific PCR product; extra peaks could mean contamination or primer-dimers.
- Lower Ct values indicate higher expression of your target gene in the sample.
- What was the Ct value for your target gene? What does that number tell you?
- Did your sample show a strong or weak gene expression level? How can you tell?
- What did the melting curve look like? Was your product specific?
- If you saw multiple peaks in the melting curve, what might that suggest?
- How might this method be useful in real-world applications like detecting disease or measuring GMO content?
- How could qPCR be used to compare gene expression across different tissues or organisms?
- If you were designing a qPCR test for a virus, what steps would you take to ensure accuracy and specificity?
- Imagine your SYBR Green fluorescence didn’t increase. List three potential reasons why and how you’d troubleshoot them.