PCR (Polymerase Chain Reaction) PDF

University of Petra Faculty of Dentistry Cellular and Molecular Biology lab PCR (Polymerase Chain Reaction) Dr. Duaa Quedan Molecular Biology lab for Dental student What is PCR? PCR stands for Polymerase Chain Reaction. It is a widely used technique in molecular biology to amplify a single or a few...

University of Petra Faculty of Dentistry Cellular and Molecular Biology lab PCR (Polymerase Chain Reaction) Dr. Duaa Quedan Molecular Biology lab for Dental student What is PCR? PCR stands for Polymerase Chain Reaction. It is a widely used technique in molecular biology to amplify a single or a few copies of a piece of DNA, generating thousands to millions of copies of a particular DNA sequence. Application of PCR 1. Research: PCR is widely used in molecular biology research for cloning, sequencing, and studying gene expression and genetic variation. 2. Medical Diagnostics: PCR is employed in medical diagnostics to detect pathogens such as bacteria and viruses, identify genetic mutations associated with diseases, and perform prenatal testing and paternity testing. 3. Forensics: PCR is used in forensic analysis to amplify DNA from crime scene samples for DNA profiling and identification of suspects. 4. Genetic Engineering: PCR is crucial in genetic engineering techniques like site-directed mutagenesis, gene cloning, and gene editing. Principles of PCR 1.Denaturation: The first step involves heating the DNA sample to around 95°C. This high temperature causes the doublestranded DNA to separate (denature) into two single strands by breaking the hydrogen bonds between the base pairs. Principles of PCR 2. Annealing: The temperature is then lowered to around 50-60°C. During this step, short DNA sequences called primers bind (anneal) to the single-stranded DNA at complementary regions adjacent to the target sequence. These primers provide the starting point for DNA synthesis. Principles of PCR 3. Extension (or elongation): The temperature is raised to around 72°C, which is the optimal temperature for the DNA polymerase enzyme to synthesize new DNA strands. The enzyme extends the primers by adding complementary nucleotides, synthesizing new DNA strands that are complementary to the template DNA. Components of PCR 1.DNA template 2.Primers 3.DNA polymerase 4.Nucleotides 5.Buffer Components of PCR: DNA Template The DNA sample containing the target sequence to be amplified. In this experiment we are using human DNA/ Target gene GAPDH Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an enzyme of about 37kDa that catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules. Components of PCR: Primer It is a short, single-stranded DNA sequences that are complementary to the sequences flanking the target region. These primers determine the specificity and efficiency of the amplification. Components of PCR: Primer Design of PCR Primers: 1.Specificity: Primers must be designed to anneal specifically to the target DNA sequence and not to any other sequences in the DNA sample. This specificity is crucial for accurate amplification. 2.Length: PCR primers typically range in length from 18 to 25 nucleotides. Shorter primers may be less specific, while longer primers may have higher specificity but lower annealing efficiency. 3.GC Content: The GC content (percentage of guanine and cytosine nucleotides) of primers should be balanced to ensure stable primer binding and efficient amplification (50-55%). 4.Melting Temperature (Tm): The Tm is the temperature at which half of the primer molecules are bound to the template DNA. The Tm of the primers should be close to each other to promote similar annealing temperatures. 5.Avoiding Self-Complementarity and Primer-Dimer Formation: Primers should not have significant self-complementarity or complementary regions with each other to prevent non-specific binding and primer-dimer formation, which can interfere with PCR amplification. Components of PCR: Primers 2 Types of primers: - Forward primer (Sense): ACC ACA GTC CAT GCC ATC AC - Reverse primer (Anti-sense): TCC ACC ACC CTG TTG CTG TA Gene of interest Components of PCR: DNA polymerase 1. Thermophilic Nature: Taq polymerase is derived from Thermus aquaticus, a bacterium that thrives in high-temperature environments, such as hot springs. Taq polymerase is stable at high temperatures, making it ideal for PCR, which involves repeated heating and cooling cycles. 2. DNA Polymerase Activity: Taq polymerase is a DNA-dependent DNA polymerase, meaning it synthesizes new DNA strands using single-stranded DNA templates and deoxyribonucleotide triphosphates (dNTPs). It adds nucleotides to the growing DNA strand in a 5' to 3' direction. 3. Heat Stability: One of the most important properties of Taq polymerase is its heat stability. It remains active through the high-temperature denaturation step of PCR (usually around 94-98°C), allowing the enzyme to withstand the repeated heating cycles required for DNA denaturation. 4. Error Rate: Taq polymerase has a relatively high error rate compared to other DNA polymerases, leading to a higher likelihood of introducing mutations during PCR. However, this property can be advantageous in certain applications, such as generating genetic diversity in directed evolution experiments. 5. Extension Temperature: The optimal temperature for the DNA synthesis activity of Taq polymerase is typically around 72°C. This is the temperature used during the extension step of PCR, where the enzyme adds nucleotides to the primers, synthesizing new DNA strands. 6. Fragment Length: Taq polymerase is suitable for amplifying DNA fragments of moderate length, typically up to a few kilobases. For longer DNA fragments, other DNA polymerases with higher fidelity and processivity may be preferred. 7. Commercial Availability: Taq polymerase is commercially available from various suppliers in purified form, often with accompanying buffers optimized for PCR. It is one of the most widely used DNA polymerases in molecular biology laboratories worldwide. Components of PCR: Nucleotides Deoxyribonucleotide triphosphates (dNTPs) are the building blocks of DNA synthesis. These are the individual units that DNA polymerase uses to add nucleotides (A, T, C, and G) to the growing DNA strand during the extension step of PCR. Each dNTP consists of a deoxyribose sugar, a phosphate group, and one of the four nitrogenous bases (adenine, thymine, cytosine, or guanine). Components of PCR: Buffer A solution containing salts and other components that provide optimal conditions for the PCR reaction, including pH (8.0 to 9.0) and ionic strength. Activity