Polymerase Chain Reaction (PCR) Lecture 21 - Vision College of Medicine

E Polymerase Chain Reaction (PCR) by Dr.Ezat Mersal Explain principles and procedures of PCR Discuss medical applications of PCR  & I  PCR,polymerase chain reaction, is an in-vitro techniq...

E Polymerase Chain Reaction (PCR) by Dr.Ezat Mersal Explain principles and procedures of PCR Discuss medical applications of PCR  & I  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 3  Reagents Needed short DNA sample which you want to amplify DNA polymerase ·  Taq DNA polymerase – Works at high temps up to 95 °C  Nucleotides  Called dNTPs (deoxynucleotide triphosphates) Pair of primers  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  Apparatus to perform about 35 cycles of a three temperature procedure 95 °C , 50-60 °C, 72 °C The DNA sample , DNA polymerase, buffer, nucleoside triphosphates, and primers are placed in a thin-walled tube and then these tubes are placed in the PCR thermal cycler 5  - As &I D It bone break Put all reagents into a PCR tube gi 1. Melting or heat denaturation double strand Break the DNA ladder down the middle to create two strands, a 5’ to 3’ strand and a 3’ to 5’ strand 2. Annealing Leading legging > - Adding pramer Bind each primer to its appropriate strand  5’ primer to the 5’ to 3’ strand leading  3’ primer to the 3’ to 5’ strand lagging 3. Extending Copy each strand  DNA polymerase 6  & go , :ei jggie Initial Melt: 94ºC for 10 minutes melting Melt: 95ºC for 30 seconds - > Anneal:& 55ºC for 30 seconds Extend: 72ºC for 1 minute > - begging Final Extension: 72ºC for 6 minutes > - the end Hold: 4ºC 7 Step 1: Denature DNA At 95C, the DNA is denatured (i.e. the two strands are separated) 8 Step 2: ° 55 C Primers AnnealA At 40C- 65C, the E primers anneal (or M O bind to) their complementary 3 primer s'primer sequences on the single strands of G DNA I 3 5 31 & is & Joe s'primer o 11g 5) 3s" = Je s'primer $1 gis 9 Step 3: ⑮ 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. S S DNA's __. 10  Gel electrophoresis detects the presence of DNA in a sample  Gelelectrophoresis 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  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  DNA fragments are then visualized in the gel with a special dye  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 Chang hig 13 20 : V Multiplex  PCR ~  Asymmetric PCR ~  Hot-start PCR W Inverse PCR  LNested PCR  ~Quantitative PCR (Q-PCR)  VReverse Transcription PCR (RT-PCR)  L  Thermal asymmetric interlaced PCR (TAIL- PCR) W Touchdown PCR (Step-down PCR)  14 – Extremely high sensitivity, may detect down to one viral genome per sample volume – Easy to set up – Fast turnaround time 15  – Extremely liable to contamination – High degree of operator skill required fules – A positive result may be difficult to interpret, especially with latent viruses such ger gige as CMV (Cytomegalovirus), where any - · seropositive person will have virus present in D their blood irrespective whether they have g i disease or not. 16  Amplification and quantification of DNA  Amplification of RNA  Diagnosis of diseases  PCR in comparison of different genomes  Drug therapy 17  Genetic disorders diagnosis as thalassemia ,CML  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.  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. 18  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 p.  GENETIC ANALYSIS AN INTEGRATED APPROACH Mark F. Sanders , John L. Bowman Second edition 2015 ISBN 978 0-321-94890-8 (student edition) www.pearsonhighered.com