L21_ PCR PDF - Polymerase Chain Reaction

# Vision Collage of Medicine ## Year 2, Level 3, Lecture 21 ### Polymerase Chain Reaction (PCR) **By Dr. Ezat Mersal** ## Outlines - Explain principles and procedures of PCR - Discuss medical applications of PCR ## Amplification Amplification is the process to increase the gene. **Polymeras...

# Vision Collage of Medicine ## Year 2, Level 3, Lecture 21 ### Polymerase Chain Reaction (PCR) **By Dr. Ezat Mersal** ## Outlines - Explain principles and procedures of PCR - Discuss medical applications of PCR ## Amplification Amplification is the process to increase the gene. **Polymerase Chain Reaction (PCR)** - PCR, polymerase chain reaction, is an in-vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence. - **Amplify** = making numerous copies of a segment of DNA. - PCR can make billions of copies of a target sequence of DNA in a few hours. ## What I need to do: Reaction Components ### Reagents Needed 1. **DNA sample:** the DNA sample which you want to amplify 2. **DNA polymerase:** enzyme to increase the number of DNA. - Taq DNA polymerase - Works at high temps up to 95°C 3. **Nucleotides:** Called dNTPs (deoxynucleotide triphosphates) 4. **Pair of primers:** for each strand - One primer binds to the 5’ end of one of the DNA strands. - The other primer binds to the 3’ end of the anti-parallel DNA strand. 5. **Apparatus:** to perform about 35 cycles of a three temperature procedure (95°C, 50-60 °C, 72 °C) ## What are the components (what I need for PCR)? 1. **DNA sample:** to amplify 2. **DNA polymerase:** Taq DNA polymeres (This works at high temperature) 3. **Nucleotides (dNTPs):** deoxynucleotid triphosphate (This is found in cells) 4. **Pair of primer:** - one attaches to the 5' end of DNA strand - other one attaches to the 3’ end of DNA strand. 5. **Apparatus:** to perform about 35 cycles of a three temperature procedure (95°C, 50-60 °C, 72 °C) (This machine provides heat). ## PCR Thermal Cycler - Put the DNA sample, DNA polymerase, buffer, nucleoside triphosphates, and primers in a thin-walled tube and then place the tubes in the PCR thermal cycler. ## Steps of PCR Thermal Cycler 1. **Initial melt:** Put the DNA sample in 94°C for 10 minutes (This is called initial melt). This weakens hydrogen bonds. 2. **Denaturing:** Increase the heat to 95°C for 30 seconds (That will break the hydrogen bonds.) So the double strands will break. 3. **Annealing:** Decrease the temperature to 55°C for allows the primer to attach the ends of DNA. (This process is called: Anneal.) 4. **Extension:** Increase the temperature to 72°C to allow the enzyme (Taq polymeres) to work for 1 minute. (Polymeres cannot work until it sees the primer, Polymeres = adding nuclitide to the strand to complete it.) 5. **Final extension:** Leave the new double strand for 6 minutes to confirm the process (The final extension). 6. **Hold:** Stop the machine at 4°C (The hold). ## PCR Protocol ### Steps 1. **Melting ** (or heat denaturation) - Break the DNA ladder down the middle to create two strands, a 5’ to 3' strand and a 3' to 5' strand. 2. **Annealing** - Bind each primer to its appropriate strand. - 5' primer to the 5' to 3' strand - 3' primer to the 3' to 5' strand 3. **Extending** - Copy each strand. - DNA polymerase ## Temperature Protocol | Step | Temp | Time | Notes | | -------- | -------- | -------- | -------- | | Initial melt | 94°C | 10 minutes | | | Melt | 95°C | 30 Seconds | | | Anneal | 55°C | 30 Seconds | Put primer | | Extend | 72°C | 1 minute | | | Final Extension | 72°C | 6 minutes | | | Hold | 4°C | | | ## The three main steps of PCR ### Step 1: Denature DNA - At 95°C, the DNA is denatured (i.e., the two strands are separated). ### Step 2: Primers Anneal - At 40°C-65°C, the primers anneal (or bind to) their complementary sequences on the single strands of DNA. ### Step 3: DNA polymerase Extends the DNA chain - At 72°C, DNA polymerase extends the DNA chain by adding nucleotides to the 3' ends of the primers. ## Gel Electrophoresis of DNA - Gel electrophoresis detects the presence of DNA in a sample. - Gel electrophoresis detects the number of nucleotides in a fragment of DNA. - e.g., the number of nucleotides in a DNA region which was amplified by PCR - I need to know the number of nucleotides that I want to amplify in PCR. ## How Gel Electrophoresis of DNA Works 1. A sample which contains fragments of DNA is forced by an electrical current through a firm gel which is really a sieve with small holes of a fixed size. * Phosphate group in DNA is negatively charged so it is moved towards a positive electrode by the current. * Longer fragments have more nucleotides. - So have a larger molecular weight. - So are bigger in size. - So aren't able to pass through the small holes in the gel and get hung up at the beginning of the gel. * Shorter fragments are able to pass through and move farther along the gel. * Fragments of intermediate length travel to about the middle of the gel. 2. DNA fragments are then visualized in the gel with a special dye. 3. The number of nucleotides are then estimated by comparing it to a known sample of DNA fragments which is run through the gel at the same time ## Electrophoresis of DNA - A diagram depicting a gel imaging device, and its components. ## PCR Types and Variations - Multiplex PCR - Asymmetric PCR - Hot-start PCR - Inverse PCR - Nested PCR - Quantitative PCR (Q-PCR) - Reverse Transcription PCR (RT-PCR) - Thermal asymmetric interlaced PCR (TAIL-PCR) - Touchdown PCR (Step-down PCR) ## Advantages of PCR - Extremely high sensitivity, may detect down to one viral genome per sample volume. - Easy to set up. - Fast turnaround time. ## Disadvantages of PCR - Extremely liable to contamination. - High degree of operator skill required. - A positive result may be difficult to interpret, especially with latent viruses such as CMV (Cytomegalovirus), where any seropositive person will have virus present in their blood irrespective whether they have disease or not. (So maybe you have the virus but it's not active, but the PCR can't detect if it's active or not.) ## Medical Applications of PCR - Amplification and quantification of DNA - Amplification of RNA - Diagnosis of diseases - PCR in comparison of different genomes - Drug therapy ## Medical Applications of PCR - Genetic disorders diagnosis as thalassemia CML (chronic myelogenous leukemia T(22;9)) - Pathogenic microorganisms, including some viruses, bacteria, parasites and fungi, cause infectious diseases and can be identified using PCR, aiding efficient diagnosis and treatment. - In some cases, gene therapy is available to address these disorders, and PCR is used to monitor the functioning of the relevant genes and gene segments (so the gene is not active so I can give the gene active to this pt). - PCR is also used in molecular diagnostics and biochemical analyses. These techniques can be used in drug development, especially in measuring the efficacy of drug therapy and research into cancer detection and treatment. ## References - Basic *Genetics: A human approach* / BSCS. Dubuque, IA, Kendall/Hunt Pub. Co., c1999. 147 p. QH431.B305 1999 - *Genes, ethnicity, and ageing*. Edited by Lincoln H. Schmitt, Leonard Freedman, Rayma Pervan. Nedlands, Australia, Centre for Human Biology, University of Western Australia ; Singapore, River Edge, NJ, World Scientific, c1995. 100 p.QH455.G45 1995 - *Genetic polymorphisms and susceptibility to disease*. Edited by M.S. Miller and M.T. Cronin. New York, Taylor & Francis, 2000. 266 р. - *Genetic analysis: AN integrated approach*. Mark F. Sanders, John L. Bowman. Second ed. 2015 ISBN 9780-321-94890-8 (student edition) www.pearsonhighered.com.