PCR Lab (1) (3) PDF

Polymerase Chain Reaction (PCR) BY  Ass. Prof. Dr. Amany Wahb Assistant Professor of Medical Biochemistry & Molecular Biology  Faculty of Medicine-Helwan University ⦿ Contents PCR concept. Applications of PCR. PCR reaction components....

Polymerase Chain Reaction (PCR) BY  Ass. Prof. Dr. Amany Wahb Assistant Professor of Medical Biochemistry & Molecular Biology  Faculty of Medicine-Helwan University ⦿ Contents PCR concept. Applications of PCR. PCR reaction components. PCR cycle. Types of PCR.  Introduction polymerase chain reaction (PCR) is an in-vitro enzymatic DNA amplification technique for amplification of a region of DNA. The amplified DNA sequence must be known or it lies between two regions of known sequence. ⦿ PCR allows the synthesis of millions of copies of a specific nucleotide sequence in a few hours. ⦿ DNA can be obtained from any source (viral, bacterial, plant, animal) 1.Genome mapping and gene function determination. 2. Biodiversity studies ( e.g. evolution studies). 3. Detection of drug resistance genes. 4. Forensic analysis of DNA samples.(Paternity test) 5. DNA fingerprinting for analysis of evidence from crime scenes using a single hair or spot of blood.  6. Diagnostics: Prenatal testing of genetic diseases Early detection of cancer (for example, screening of family members in case of genetic types of cancer. Diagnosis of viral infections (Hepatitis B, C, AIDS, COVID19,…ETC) Diagnosis of genetic disorders: sickle cell anemia,…etc Template DNA: a sample of the DNA that you wish to replicate. Two Primers (forward and reverse): Are starting/ending points for DNA replication; thus define the region of DNA to be replicated in PCR. Primers range from 15 to 30 nucleotides, Synthetically produced, complimentary to the 3’ ends of target DNA, not complimentary to each other).  DNA polymerase: (Enzyme that replicates DNA). The most commonly used is the Taq DNA polymerase (Thermostable DNA polymerase which is derived from Thermus aquatics). dNTPs (DeoxyribonucleosideTriphosphates) (dATP, dCTP, dGTP, dTTP). Mg2+ : Essential co-factor of DNA polymerase. 1. Denaturation of DNA The target DNA to be amplified, is heated to 95°C to separate dsDNA to single strands. 2. Annealing of Primers A Primer is a single stranded oligonucleotide molecule, complementary to sequences surrounding the target sequence. Two primers are required for PCR reactions. During the annealing phase, DNA is cooled to 50°C& the 2 primers (one for each strand) anneal to complementary sequence on the ssDNA. 3. Extension of Primers DNA polymerase uses dNTPs in excess to initiate the synthesis of 2 strands complementary to the original ones. The temperature is adjusted to 72°C in this phase. 1. Which of the following is not a thermostable polymerase? (a) pfu polymerase (b) Taq polymerase (c) Vent polymerase (d) DNA polymerase III 2. Which of the following is the basic requirement of PCR reaction? (a) Two oligonucleotide primers (b) DNA segment to be amplified (c) A heat-stable DNA polymerase (d) All of the above 3. Which of the following is the first step in the polymerase chain reaction? (a) Annealing (b) Extension (c) Denaturation (d) Primer binding A programmable device that cycles between specific temperatures for set periods of time – used for PCR. ⦿ 20-30 cycles are run, amplifying DNA by a million- billion fold “exponential amplification”. ⦿ A special DNA polymerase must be used that can withstand high temperatures of the reaction. Thermus acquaticus bacterial Taq Polymerase. ⦿ Resulting PCR products are separated by gel electrophoresis & visualized by UV transilluminator. 1- Enumerate requirment for PCR technique ? 2- Enumerate three steps of PCR? 3- What is the name of this instrument? 4- What is the technique ? 1. Denaturation is the process of _________. (a) Heating between 72°C (b) Heating between 40 to 60°C (c) Heating between 90 to 98°C (d) None of the above 2. Polymerase used for PCR is extracted from -----------. (a) Homo sapiens (b) Thermus aquaticus (c) Escherichia coli (d) Saccharomyces cerevisiae 3. How many DNA duplexes are obtained from one DNA duplex after 5 cycles of PCR? (a) 8 (b) 4 (c) 32 (d) 16  Reference Harvey, Richard A., Ph. D. (2017). Lippincott's illustrated reviews: Biochemistry. Philadelphia :Wolters Kluwer Health, 7th edition 32 33