Instruction 3 - PCR

DNA and RNA Replication - PCR Reaction

• PCR (Polymerase Chain Reaction) is a technique for selectively amplifying DNA in a laboratory setting. It mimics the natural DNA replication process.
• PCR allows for the duplication of DNA sequences ranging from a few hundred to several thousand nucleotides.
• The PCR method was developed in 1987 by scientists at Cetus Corporation in the USA.
• Kary Mullis received the Nobel Prize in Chemistry in 1993 for his work on the foundation of PCR.
• PCR enables selective in vitro amplification of DNA by mimicking in vivo DNA replication.

Purposes of PCR Reactions

• PCR can be used to amplify a specific sequence of DNA from a sample.
• Applications include gene expression analysis, diagnostics, mutation detection, pathogen identification, and forensic science.
• PCR is also used for genetic engineering, gene cloning, functional gene analysis, and organism modification.
• PCR allows for the duplication of any DNA sequence ranging from a few hundred to several thousand nucleotides in length.

Thermal Cycler

• PCR reactions are conducted in a thermal cycler, which programs temperature changes and reaction durations.
• The heart of the thermal cycler is an aluminum block coupled with a Peltier element. This arrangement allows for rapid temperature changes of many degrees within a few seconds.
• In the thermal cycler, the temperature and duration of individual reaction stages and the number of cycles are programmed.

PCR Cycle

• The PCR reaction consists of three main stages, repeated 30-40 times:
  • Denaturation: DNA is heated (92-96°C) to break the hydrogen bonds, separating the DNA strands.
  • Annealing: The mixture is cooled (37-72°C) and primers anneal to complementary regions on the DNA template. Primers are short, single-stranded sequences that target specific regions of the DNA being amplified.
  • Extension: The temperature is raised (68-75°C), and DNA polymerase extends the primers by adding nucleotides to build new complementary strands.

Denaturation

• During denaturation, DNA is heated to break the hydrogen bonds. Initial denaturation is typically sufficient for genomic DNA.

Annealing

• The mixture is cooled and specific primers bind to the single-stranded DNA sequences. The temperature required for annealing depends on the primer sequences.

Elongation

• DNA polymerase adds nucleotides to the primers. The optimal temperature for elongation is typically 72-75°C. The rate of nucleotide addition (DNA synthesis) is approximately 1000 nucleotides per minute.

PCR Cycle Thermal Profile

• A graph displays the temperature profile for PCR cycles, with steps for denaturation, annealing, and extension. A digital display/machine graphically visualizes the cycles.
• The graph shows the temperature and times for each step in a PCR cycle.

The Number of DNA Amplification Cycles

• The number of PCR cycles needed depends on the amount of initial template DNA and the desired amount of product. Fewer cycles are appropriate with lower starting amounts. Higher initial amounts require fewer cycles. The theoretical yield of double-stranded molecules after "n" cycles is 2^n.

Exponential DNA Amplification

• The visualization demonstrates the exponential increase in the number of DNA copies during PCR. The amount of copies doubles with each cycle.
• The graph shows the exponential growth of DNA copies during PCR cycles

Components of the PCR Mixture

• Template DNA: The DNA sequence to be amplified.
• Primers: Short, single-stranded DNA segments that are complementary to the ends of the target sequence.
• DNA Polymerase: Typically a heat-stable enzyme (e.g., Taq polymerase) that adds nucleotides to the growing DNA strands.
• dNTPs (Deoxynucleotide triphosphates): The building blocks of DNA (e.g., dATP, dTTP, dCTP, dGTP).
• Buffer: Helps maintain the optimal pH and contains magnesium ions (Mg²⁺).
• Water: Nuclease-free water to dissolve components.

PCR Efficiency

• Factors influencing PCR efficiency include:
  • Annealing temperature
  • Primer concentration/template DNA concentration (quantity, purity)
  • MgCl2 concentration
  • dNTP concentration (e.g., dATP, dTTP, dCTP, dGTP)
  • Polymerase concentration
  • Primer extension temperature
  • Reaction buffer

Appropriate Annealing Temperature

• The optimal annealing temperature is based on the matching between the primers and their complement on the template DNA.

Annealing Temperature - Too High/Too Low

• If the annealing temperature is too high, the primers will not bind to the template.
• If the annealing temperature is too low, non-specific products can form.

Gradient PCR

• Gradient PCR is a method for optimizing the annealing temperature for a specific primer pair. The results can show the optimal annealing temperature on a graph.
• By using a temperature gradient in the thermal cycler, we can determine the ideal annealing temperature.

PCR Reaction Optimization

• PCR reactions often do not have 100% efficiency. Reaction conditions may need adjusting to improve its efficiency.
• Reaction conditions must be adjusted to improve PCR efficiency when it isn't 100 percent.

Concentration of Template and Primers

• The optimal concentration of template DNA and primers vary based on the reaction type (e.g., genomic, plasmid). Too high or too low concentrations can negatively impact amplification efficiency by increasing non-specific products.

Divalent Ions

• Magnesium ions (Mg²⁺) are crucial for DNA polymerase activity. The concentration needs to exceed the number of phosphate groups in the reaction mixture.

Deoxynucleotides (dNTPs)

• The optimal concentration of dNTPs is 200-400 μM. An unequal ratio of dNTPs negatively impacts PCR efficiency.

DNA Polymerase (e.g., Taq)

• Heat stability: Taq polymerase is stable at high temperatures, enabling multiple cycles of denaturation without degradation.
• 5' to 3' synthesis: The enzyme adds nucleotides to the growing DNA strand.
• No 3'-5' proofreading: Taq polymerase has a low error rate of adding wrong nucleotides and lacks the proofreading ability.

Taq Polymerase Concentration

• 1 unit of Taq enzyme / reaction is commonly sufficient. Higher concentrations can produce non-specific products.

Elongation Temperature

• Elongation temperature (typically 72°C) influences the elongation rate, which depends on the specific polymerase and can vary from 1 kb/min to several kb/min.

Reaction Buffer

• The buffer is specific to a polymerase and stabilizes the pH of the reaction mixture. Monovalent potassium chloride (KCI) ions are important components, with standard concentrations aiding with the amplification of larger fragments. Higher concentrations improve amplification for shorter fragments (<500 base pairs).

Reaction Mixture Additives

• Additives like Bovine Serum Albumin (BSA), ammonium sulfate, and Triton can stabilize the polymerase and modify interactions between the template and primers.

Risks

• False positive: Post-amplification product contamination, reagent contamination, contamination from organisms,
• False negative: Inhibitors of the reaction, incorrect sample volume, poor quality of isolated nucleic acids, equipment malfunction (e.g., thermocycler), issues related to reagents (e.g., magnesium concentration, primer synthesis).

Necessary Control Reactions

• Positive control: Uses a known DNA template to ensure the reaction is working correctly. Absence of the product indicates an issue with the reaction or its preparation.
• Negative control: Uses water as a template; the presence of a product indicates contamination. Absence of a product confirms that components were not accidentally contaminated with the DNA template.

Varieties of PCR

• End-point PCR: Quantifies DNA amplification qualitatively by gel electrophoresis.
• Multiplex PCR: Amplifies multiple DNA regions simultaneously in one tube using different primers.
• Real-time PCR (qPCR): Quantifies DNA amplification in real-time using fluorescence signals; no gel electrophoresis is required.

End-point PCR vs. Real-Time PCR

• End-point PCR: Qualitative analysis of PCR products for later diagnostic or preparation steps.
• Real-time PCR: Quantitative analysis enabling quantification of the initial amount of a specific DNA sequence.

Real-Time PCR (PCR in real-time)

• Uses fluorescent dyes like SYBR Green to monitor the accumulation of DNA product in real time; no gel electrophoresis is needed for analysis.

Reverse Transcriptase PCR (RT-PCR)

• Used to analyze mRNA. Conversion of mRNA into cDNA before PCR amplification is necessary for stability.
• cDNA is analyzed by quantitative real-time PCR (qPCR).

DNA Electrophoresis

• Used to separate DNA molecules based on their size and charge.

Electrophoresis

• An analytical method separating charged molecules (proteins, DNA) in an electric field. Movement occurs based on molecule size and charge under constant pH.

DNA and Protein Electrophoresis

• DNA and proteins migrate in different directions in an electric field due to different charges; effective separation needs a constant pH.

Electrophoretic Carrier

• For better resolution, stability, and repeatability in separating molecules, electrophoretic carriers are used. Agarose and polyacrylamide are examples.

Agarose Gels

• Prepared from a seaweed extract. Varying concentrations create different pore sizes. Useful for separating DNA fragments from 100 to 40,000 base pairs.

Agarose Gel Electrophoresis

• Advantages: Wide range of DNA separations; easy preparation; non-toxic
• Disadvantages: Low mechanical strength; limited resolving power

Polyacrylamide Gels

• Prepared from acrylamide monomers and cross-linking substances. Adjusting concentrations allows for controlled degrees of cross-linking and pore sizes.

Polyacrylamide Gel Electrophoresis

• Enables separation of protein molecules with molecular weights from 5 kDa to 300 kDa and polynucleotides ranging from 5 to 2,000 base pairs.

Electrophoresis Chambers

• Vertical (common for proteins), horizontal (often for DNA), and capillary electrophoresis are various chamber types.

Zone Electrophoresis

• The most common electrophoresis method. Separates components in a carrier under constant pH by using the different electrophoretic mobility characteristics.

DNA Migration in Agarose Gel

• Migration rate is inversely proportional to the log10 of its molecular weight. Linear, open circular, and supercoiled circular DNA forms migrate at different rates.

DNA Electrophoresis Process

• Sample DNA or proteins are mixed with a loading buffer and dye into a gel to be separated under an electric field.

Staining

• Visualization of separated molecules is necessary and involves staining with dyes (e.g., Coomassie brilliant blue, silver staining, fluorescent dyes).

Dyes

• Coomassie Brilliant Blue/Amido Black: Stain proteins; allow detection of 1 µg of protein.
• Silver staining: Stain proteins and nucleic acids; allows detection of 10 ng of protein or DNA.
• Fluorescent dyes (e.g., Ethidium Bromide, Midori Green): Usually stain DNA for visualization under UV light; vary in toxicity and detection sensitivity.

Documentation of Electrophoretic Separations

• Polyacrylamide gels can be dried using filter paper for documentation. Dried samples can be stored. Agarose gels are typically photographed.

Gel Electrophoresis Simulation

• Visualization of DNA/protein separation patterns in different simulated gel electrophoresis results. Displays different gel concentrations and times for separation.

Test your knowledge on the principles and procedures of Polymerase Chain Reaction (PCR). This quiz covers nucleotide addition rates, primer extension, cycle recommendations, and common components of PCR mixtures. Perfect for students and professionals looking to assess their understanding of molecular biology techniques.