Lab 10 Polymerase Chain Reaction (PCR) Amplification of PTC Gene PDF

Lab 10 Polymerase Chain Reaction (PCR) & Amplification of PTC Gene  DNA Amplification By Polymerase Chain Reaction (PCR) PCR, established in 1984, is a powerful method for rapidly amplifying specific DNA segments in the laboratory. This sensitive technique can produ...

Lab 10 Polymerase Chain Reaction (PCR) & Amplification of PTC Gene  DNA Amplification By Polymerase Chain Reaction (PCR) PCR, established in 1984, is a powerful method for rapidly amplifying specific DNA segments in the laboratory. This sensitive technique can produce billions of copies of a target DNA sequence from a small template in just a few hours. Amplify = making numerous copies of small specific DNA segment. 2 PCR is now an essential tool of Molecular Biology.  What are Needed for PCR? 1. DNA template, that contains the target sequence you want to copy and amplify. 2. Primers, two short, single-stranded DNA molecules that serve as primers which flank the target DNA sequence. 3. All Deoxyribose Nucleotides Triphosphates (dNTPs); (dATP, dCTP, dGTP, dTTP) which, are the building blocks for the new DNA copies. MgCl2 4. Taq polymerase enzyme, which is heat stable polymerase enzyme, that is isolated from the bacteria, Thermus aquaticus which live in hot springs. 5. Buffer to maintain the suitable pH (alkaline). 6. Magnesium Chloride, MgCl2 (enzyme cofactor, catalyst). 7. Thin-walled tubes very sensitive to temperature changes. 8. Thermal cycler (a device that can change the temperature dramatically in a very short 3 period of time).  The Essential Three Steps of PCR DNA Replication Step 1: Denaturation 3’ 5 7 (Separation) 5’ 5’ 3’ 2 3’ 5’ Ligase 8 5’ 3’ 5’ 3’ 5’ 3’ 1 ≈ 95oC 4 3 3’ 3’ 5’ Exonuclease 6 Denaturation or separation of the two https://www.youtube.com/ 4 watch?v=TNKWgcFPHqw DNA strands that occurs at about 95ºC.  Step 2: Primer Annealing (Binding) ≈ 50oC 5’ 3’ 3’ 5’ Reverse Primer Forward Primer 5’ 3’ 3’ 5’ Primers anneal or bind to the complimentary sequence on the target DNA sequence at about 50oC. Primers are chosen such that one is complimentary to the first template strand at the first 3’ end of the target sequence and the second is complimentary to the second template strand at the opposite 3’ end of the target 5 sequence.  Step 3: Primer Extension (Elongation) ≈ 72oC 5’ 5’ 3’ 3’ 5’ 3’ 5’ Extension 3’ 5’ Extension 5’ 3’ 5’ 3’ 3’ 5’ Extension or elongation of the new DNA strands which synthesized by Taq DNA polymerase enzyme that starts at the primers 3’ OH ends to extend from 5’→ 3’ direction by addition of the complimentary deoxyribose nucleotides triphosphates (dNTPs) (A = T & C G). 6 The extension or DNA elongation occurs at about 72oC.  The successive repeated cycles will begin by denaturing the new DNA strands in each cycle, annealing, extension and so on.  PCR Exponential Amplification Exponential amplification means increasing of the number of copies during the PCR progress. Calculation of the exponential amplification copy number by raising 2 to the power of the PCR Cycle Number. Exponential Amplification = (2)PCR Cycle Number e.g., PCR cycle number 35 = 235 = 34 billion copies. 8  PCR is a Very Powerful Tool in Molecular Genetics 1.PCR can be used to identify the genotype possibility of an individual who has a particular https://www.youtu be.com/watch?v= DbR9xMXuK7c https://www.publichealthontario.ca/en/diseases-and- conditions/infectious-diseases/respiratory-diseases/novel- coronavirus/lab-testing-ontario/covid-19-pcr-testing alleles of a certain gene as, PTC gene genotypes which will be identified using PCR. 2.Some genetics diseases can be diagnosed by PCR tool, such as: Cystic fibrosis, CF (cc) of homozygous recessive, and Covid 19 virus. 3.PCR is used in forensic medicine starting with a single drop of blood or piece of hair in the crime seen and amplifying those DNAs sufficiently to allow for DNA electrophoresis and identification of the true suspect. 4.PCR can amplify fragments of interest in an individual's DNA by choosing number of primers giving us a unique “DNA Fingerprint” that can be used to identify an individual identity, for example 9 paternity case or if baby changed in hospitals. Can You Taste Phenylthiocarbamide (PTC)? Modern biological research merges genetics, biochemistry, and bioinformatics. Taste TAS2R38 gene receptors is a small protein receptor with 1 exon (intron less) gene of 1002 nucleotides & 333 amino acids. PTC tasting is fully cloned and inherited as a dominant trait. Each student define his tasting phenotype and estimates his genotype at last. Using the PTC powder strips all students record their phenotypes of tasting ability as: strong taster, moderate taster, or nontaster. Phenotype PTC Testing. Blakeslee,1938 http://learn.genetics.utah.edu/content/begin/traits/ptc/images/DSC00103.JPG http://www.connecticutvalleybiological.com/images/br1040.jpg  Can You Taste Phenylthiocarbamide (PTC)? Single nucleotide polymorphisms (SNPs) is the DNA sequence variation occurring when a single nucleotide changed in the genome. There are 3 SNPs in the TAS2R38 gene which form a haplotype that correlates with bitter tasting. Each of the 3 changes also produces a change in the amino acids sequence of the receptor protein as shown below. Nucleotide Change Codon Change Amino Acid Change Nucleotide Position (Taster > Nontaster) (Taster > Nontaster) (Taster > Nontaster) 145 C>G CCA > GCA Proline > Alanine 785 C>T GCT > GTT Alanine > Valine 886 G>A GTC > ATC Valine > Isoleucine An assay to estimate the SNP at position 145, which has the https://www.m acromoltek.co m/utilities/cod highest correlation to tasting of the 3 polymorphisms (SNPs). on2sequence/ Students extract DNA from their cheek cells by a simple mouth swap and amplify a region of the TAS2R38 gene using PCR. The amplified fragment (amplicon) is then cut by the restriction enzyme HaeIII, which its recognition sequence GGCC includes the SNP (C/G 145) as we will be discussed in restriction enzyme Lab.  Taste PTC Results PCR amplification and then restriction cut (digestion) to identify the C/G polymorphism (SNP) in the TAS2R38 gene. The “C” taster allele is digested by HaeIII and correlates with PTC tasting. While “G” non-taster allele neither recognized nor digested.  Optimizing PCR Protocol While PCR is a very powerful technique. It is not possible to achieve the optimal PCR result without optimizing the PCR protocol trough the following critical PCR parameters: Critical PCR Parameters: 1. Concentrations of the following PCR constituents: DNA template (1ng/µl–1µg/µl). Primers (final concentration 0.1–5.0 µM of each primer). Deoxyribose Nucleotides Triphosphates (dNTPs) final concentration (200 µM of each dNTP). Taq DNA Polymerase, at a concentration of 25 units/ml (1.25 units/50 μl reaction). Magnesium Chloride catalyst of DNA polymerase (1.5–2.0 mM). 14 2. Primers Design and Primers Melting Temperature (Tm). 2. Primers Design and Primers Melting Temperature A. Primers Design: Specificity and Length: Primer annealing temperature and primer specificity are mainly dependent on primer length and primer design. Primer length is directly proportional to annealing specificity. Generally, the longer the primer, the more specific annealing. Primers between 30 – 50 bases are highly complimentary specific. B. Primers Melting Temperature (Tm): https://tmcalculator.neb.com/#!/main The aim, is to design a primer with annealing temperature between about 30 – 65°C. The relationship between annealing temperature and melting temperature is one of the important limiting factors of PCR The melting temperatures (Tm) of primers are calculated using this formula: Melting Temperature: Tm = 4 ( # C + # G ) + 2 ( # A + # T ) = °C A General Rule: Annealing Temperature = Melting Temperature – 5 = °C Both primers should be designed in such that they have similar or close melting temperatures to make the PCR working properly. 15  DNA Amplification Protocol by PCR 1.Obtain a PCR tube label with your assigned name. 2.Use a micropipette with a new tip to add 40.0 μL of PTC primer/PCR master mixture, and nuclease-free water to the tube. 3.Use a micropipette with a new tip add 10.0 μL of your cheek cell DNA directly into the primer/PCR master mixture into PCR tube. 4.Store your sample on ice until your class is ready to begin thermal cycling. 5.Place your PCR tube, along with other students' samples, in the thermal cycler that has been programmed to the following profile for 35 cycles. 6.The profile will be linked to a 4°C holding program after cycling is completed. Denaturation step: 95°C for 30 seconds. Annealing step: 50°C for 30 seconds. Extension step: 72°C for 60 seconds. 6.After cycling, store the amplified DNA on ice or at –20°C until you are ready to continue with the next Part of PCR digestion by HaeIII enzyme  PCR Protocol Thaw the freeze PCR Master Mix at room temperature. Vortex the Master Mix and then spin it briefly in a microcentrifuge to collect the material at the bottom of the tube. Keep all reagents in ice. Composition PCR Master Mix: TaqDNA Polymerase [in buffer (pH 8.5)]. Deoxyribose nucleotide Triphosphates: dATP, dGTP, dCTP, dTTP. MgCl2 Components for a 50.0 µl Volume Reaction Volume: PCR Master Mix - 2 X 25.0 µl Forward Primer - 5 µM 4.0 µl Reverse Primer - 5 µM 4.0 µl Nuclease-Free Water 7.0 µl 17 DNA Template, - 1 µg 10.0 µl http://www.youtube.com/watch?v=DkT6XHWne6E  General Steps of PCR Reaction Protocol A. Initial Denaturation: Generally, initial denaturation step at 95°C for 5 min. B. Cycles Number: Generally, 35 cycles repetition result in optimal amplification of desired products. C. The three essential PCR steps are repeated for 35 cycle: 1. Denaturation: Denaturation segment will be at 95°C for 30 sec. 2. Annealing: The annealing step is typically at 50°C for 30 sec. 3. Extension: The extension reaction is will be at 72°C for 60 sec. D. Final Extension: A final extension at 72°C for 5 min. E. Refrigeration: The thermal cycler has last refrigeration cycle, the cycling reaction can be programmed to end by holding the tubes at 4°C for 24 h or more (∞) until the restriction enzyme cut Lab. 18 C:\Users\acer1\Desktop\DNA pictures\IMG_1286.JPG PCR Thermal Cycler https://www.youtube.com/watch?v=QYpX94prb0A https://www.youtube.com/watch?v=MxDgPFNjkbw