Quick Learn: Polymerase Chain Reaction

Steps in PCR process:

1. Denaturation:

• The DNA sample is heated to around 94-98°C, causing the double-stranded DNA to separate (denature) into two single strands.

1. Primer Annealing:

• The reaction temperature is lowered to about 50-65°C. Short DNA primers, designed to match sequences on each side of the target DNA region, bind (anneal) to the single-stranded DNA.

1. Extension (Elongation):

• The temperature is raised to around 72°C. DNA polymerase enzyme synthesizes a new DNA strand complementary to the template strand by adding nucleotides to the 3' end of the primers.

1. Repeat Cycles:

• The process is repeated multiple times (usually 20-40 cycles) to amplify the target DNA exponentially. Each cycle results in a doubling of the target DNA.

After several cycles, you'll have a significant amount of the specific DNA fragment you're interested in. PCR is a crucial technique in molecular biology for tasks like DNA sequencing, diagnostics, and genetic research.

PCR Poisons

PCR poisons, in the context of polymerase chain reaction (PCR), refer to substances that can inhibit or interfere with the PCR reaction. These can lead to inaccurate or failed amplification of the target DNA. Common PCR poisons include:

1. Inhibitory Substances:

• PCR inhibitors can be present in the DNA sample itself. These include contaminants such as salts, proteins, polysaccharides, and other organic molecules that can interfere with the PCR reaction components.

1. Residual Phenol/Chloroform:

• If DNA extraction involves phenol or chloroform, residues of these substances can inhibit PCR. It's crucial to ensure complete removal during the purification process.

1. Heme:

• Blood samples may contain heme, a component of hemoglobin, which can inhibit PCR. Proper DNA extraction methods are essential to eliminate heme contamination.

1. EDTA (Ethylenediaminetetraacetic Acid):

• EDTA is a common anticoagulant in blood collection tubes. Its presence in DNA samples can inhibit PCR by chelating magnesium ions, which are essential for DNA polymerase activity.

1. Proteinase K Inhibitors:

• Proteinase K is often used in DNA extraction protocols. Inhibitors of this enzyme, if not fully removed, can affect PCR performance.

To minimize PCR inhibition, it's important to optimize DNA extraction methods, use purified DNA, and consider the use of DNA purification kits. Additionally, careful handling and storage of reagents to avoid contamination can contribute to successful PCR reactions.

Heme, a component of hemoglobin found in blood, can inhibit PCR activity by interfering with the DNA amplification process. The inhibition is primarily attributed to the presence of iron within the heme molecule. Here's how heme inhibits PCR:

1. Chelation of Magnesium Ions:

• Magnesium ions (Mg^2+) are essential cofactors for the activity of DNA polymerase, the enzyme responsible for synthesizing the new DNA strand during PCR. Heme has a strong affinity for magnesium ions and can chelate or bind to them, reducing the availability of free magnesium ions in the reaction.

1. DNA Polymerase Inhibition:

• DNA polymerase requires magnesium ions for its catalytic activity. When heme sequesters magnesium ions, it interferes with the ability of DNA polymerase to function optimally. This can lead to a decrease in the efficiency of DNA synthesis during the extension phase of PCR.

1. PCR Amplification Failure:

• Reduced magnesium ion availability affects the fidelity and efficiency of DNA polymerase, ultimately hindering the amplification of the target DNA sequence. This inhibition can result in partial or complete PCR failure, where the desired DNA fragment is not adequately replicated.

To overcome heme inhibition, it's crucial to employ effective DNA purification methods during sample preparation. Removing heme and other contaminants ensures that the PCR reaction components remain free from substances that could interfere with the enzymatic activity necessary for successful DNA amplification.

How to remove phenol/chloroform?

Complete removal of traces of phenol and chloroform from DNA samples is crucial to prevent PCR inhibition. Here are steps to ensure thorough removal:

1. Organic Extraction:

• After phenol/chloroform extraction of DNA, perform an additional step of organic extraction using a solvent like chloroform. This helps in further separating contaminants from the DNA.

1. Precipitation with Ethanol:

• Precipitate the DNA by adding cold ethanol. This step helps in concentrating the DNA and removing any remaining traces of phenol or chloroform.

1. Wash with Ethanol:

• Wash the DNA pellet with 70% ethanol to remove salts, proteins, and residual phenol or chloroform. Centrifuge the sample and carefully remove the ethanol.

1. Air-Dry or Vacuum Dry:

• Allow the DNA pellet to air-dry or use a vacuum dryer to remove any remaining traces of ethanol. Ensure that the pellet is completely dry before resuspending it in a suitable buffer or water.

1. Use Commercial DNA Cleanup Kits:

• Utilize commercial DNA cleanup kits that are designed to efficiently remove contaminants. These kits often include silica-based columns or magnetic beads for purification, providing a reliable method for eliminating impurities.

1. Check Purity with Spectrophotometry:

• Measure the DNA concentration and purity using a spectrophotometer. This helps in assessing the success of the purification process. Pure DNA should have A260/A280 and A260/A230 ratios within acceptable ranges.

1. Quality Control:

• Run a small aliquot of the purified DNA on an agarose gel to visually inspect the integrity and size of the DNA fragments. A clean, high-molecular-weight band indicates successful removal of contaminants.

1. PCR Negative Control:

• Include a PCR negative control using the purified DNA to ensure that there is no contamination affecting the PCR reaction.

By carefully following these steps and using quality purification methods, you can minimize the risk of PCR inhibition due to residual phenol or chloroform in your DNA samples.

PCR negative control

A PCR negative control, which typically involves using PCR-grade water or a DNA sample known to be free of the target sequence, is an essential component to monitor for contamination and assess the specificity of your PCR reaction. Here's how to use a PCR negative control:

1. Designating the Negative Control:

• Decide on the appropriate negative control for your experiment. This can be PCR-grade water, a mock DNA sample lacking the target sequence, or any other suitable negative control that ensures the absence of the DNA you are amplifying.

1. Setting Up PCR Reactions:

• Prepare your PCR reactions as usual, including your template DNA for amplification. In a separate reaction tube, set up the negative control using the chosen negative control material instead of template DNA.

1. PCR Cycling:

• Run your PCR program, including denaturation, primer annealing, and extension steps. The negative control reaction should go through the same cycling conditions as your experimental samples.

1. Gel Electrophoresis:

• After PCR, analyze the products using gel electrophoresis. Load a small volume of each PCR reaction, including the negative control, onto an agarose gel.

1. Interpreting Results:

• Examine the gel for the presence of bands. The absence of a band in the negative control lane indicates that there was no contamination in the reagents or during the setup process.

1. Troubleshooting:

• If the negative control shows a band, it suggests contamination in your PCR reaction. Investigate and address potential sources of contamination, such as pipettes, tubes, or reagents.

1. Repeat if Necessary:

• If contamination is suspected, repeat the PCR with a fresh negative control. Ensure all equipment and reagents are sterile, and take precautions to prevent contamination during the setup.

Using a PCR negative control is a good practice to validate the specificity of your reaction and identify potential contamination issues. It serves as a baseline to compare with the experimental samples, helping ensure the reliability of your PCR results.