PCR, Gel Electrophoresis Exercise 3 PDF

DNA and RNA Replication PCR Reaction Department of Molecular Biology and Genetics Molecular Biology  PCR reaction The Polymerase Chain Reaction (PCR) technique enables selective in vitro amplification of DNA by mimicking the phenomenon of in vivo DNA...

DNA and RNA Replication PCR Reaction Department of Molecular Biology and Genetics Molecular Biology  PCR reaction The Polymerase Chain Reaction (PCR) technique enables selective in vitro amplification of DNA by mimicking the phenomenon of in vivo DNA replication. It allows the duplication of any DNA sequence ranging from a few hundred to several thousand nucleotides in length. The PCR method was developed in 1987 by a group of scientists at Cetus Corporation in the USA. American biochemist Kary Mullis received the Nobel Prize in Chemistry in 1993 for his work on the foundation of this method.  Purposes of PCR reactions DNA amplification Detection of a specific DNA sequence in a sample. - gene expression analysis, - diagnostics, mutation detection - pathogen identification, forensic science. Copying a specific DNA sequence (gene) for: - genetic engineering, - gene cloning, - functional gene analysis - organism modification.  Thermal cycler The reaction is conducted in a specialized apparatus called a thermal cycler, where temperature and the duration of individual reaction stages, as well as the number of cycles, are programmed. The heart of the thermal cycler is an aluminum block coupled with a Peltier element, allowing very rapid temperature changes of dozens of degrees within several seconds.  PCR cycle The PCR reaction consists of three stages, which are repeated 30 to 40 times: Denaturation of DNA (30 seconds to 5 minutes; 92-96°C). Annealing of primers to the complementary region of the template (15 seconds to 1 minute; 37-72°C). Extension of primers (DNA synthesis) (68-75°C).  Denaturation During denaturation, DNA is heated, breaking the hydrogen bonds between the strands, resulting in the DNA molecule separating into two strands. The temperature and duration of the denaturation step depend on the type of template DNA used in the reaction. Initial denaturation is sufficient to denature genomic DNA.  Annealing The mixture is cooled to a specific temperature at which primers can form double-stranded hybrid structures at the "ends" of each DNA strand, specifically at the ends of the DNA fragment to be replicated. Each of the primers binds to only one strand of the target DNA.  Elongation The temperature and duration of primer elongation depend on the type of polymerase used. The optimal temperature is typically 72-75°C. The theoretical rate of nucleotide addition (DNA synthesis) is 1000 nt/min. DNA polymerase copies each single-stranded template by extending each primer in the 5' to 3' direction.  PCR cycle thermal profile 1 2 3 1 2 3  The Number of DNA Amplification Cycles The number of PCR cycles depends on the amount of template DNA in the reaction mixture and the expected PCR product yield. For fewer than 10 copies of template DNA, it is advisable to perform 40 cycles. If the initial amount of template DNA is higher, 25-35 cycles are sufficient. The theoretical yield of the reaction after "n" cycles is 2 n double-stranded, specific DNA molecules.  Exponential DNA Amplification Cycles Number of DNA chains 1 2 2 4 3 8 4 16 5 32 6 64 20 1.048.576  Components of the PCR Mixture To conduct a PCR reaction, a reaction mixture is required, which includes: Template DNA A pair of specific primers - short, single-stranded DNA segments that complementarily bind to the ends of a specific DNA sequence. DNA polymerase enzyme, stable at high temperatures. Deoxyribonucleotide triphosphates (dNTPs) - synthetic nucleotides. Buffer (containing Mg2+) Nuclease-free water.  PCR Efficiency Factors influencing PCR efficiency include: Annealing temperature Primer concentration/template DNA concentration (quantity, purity) MgCl2 concentration. dNTP concentration (dATP, dTTP, dCTP, dGTP). Polymerase (Taq) concentration Primer extension temperature Reaction buffer  Appropriate annealing temperature … 5’ 3’ Primer forward (OH) 3’ 5’ (P) 100% primer matching to the template sequence at the optimal annealing temperature (P) 5’ 3’ (OH) 3’ Primer reverse 5’ 5’ 3’ DNA polymerase Primer forward 3’ Primer reverse 5’ Annealing temperature too high … 5’ 3’ Primer forward (OH) 3’ 5’ No primer binding to the template sequence at too high annealing temperatur 5’ 3’ (OH) 3’ Primer reverse 5’  Annealing temperature too low … 3’ 5’ Primer forward (OH) 3’ 100% primer matching to the template 5’ sequence at the optimal annealing temperature 5’ 3’ (OH) 3’ Primer reverse 5’ 3’ Primer reverse 5’ 60% matching at a lower annealing temperature 5’ 3’ Primer forward 5’ Primer forward 3’ DNA polymerase 3’ Primer reverse 5’ 3’ Primer reverse 5’ Specific product Non-specific product  Gradient PCR - optimization of annealing temperature Too high annealing temperature reduces PCR efficiency. Too low annealing temperature results in the appearance of nonspecific PCR products  PCR Reaction Optimization PCR reactions typically do not proceed with 100% efficiency. To increase efficiency, it is necessary to appropriately adjust and modify reaction conditions. Many different factors can affect the effectiveness of DNA amplification by the PCR method.  Concentration of template and primers The amount of template DNA should fall within the range of: plasmid or phage DNA: 0,01 – 1 ng, genomic DNA: 0,1 – 1 µg. A higher amount of template DNA increases the content of non-specific PCR products. The concentration of each primer in the reaction should be in the range of 0.05 - 1 μM, with an optimal range of 0.2 - 0.3 μM. Too low a concentration limits reaction efficiency, while too high a concentration leads to non-specific PCR products.  Divalent ions All polymerases require divalent cations for their activity, usually magnesium ions (Mg2+). Some polymerases also require manganese ions (Mn2+). The number of Mg2+ ions must exceed the number of phosphate groups present in the reaction mixture because both free nucleotides and primers bind to Mg2+ ions. The final concentration of Mg2+ typically ranges from 0.5 to 5 mM, with an optimal range of 1.5 - 2.0 mM for most polymerases.  Deoxynucleotides (dNTPs) The used dNTP mix should contain equal amounts of the four nucleotides: dATP, dTTP, dCTP, and dGTP. An unequal amount of dNTPs reduces amplification efficiency. The optimal concentration of dNTPs is 200 - 400 μM. Excessive concentration can chelate the Mg2+ ions required by the polymerase. Low concentration increases DNA replication accuracy but reduces efficiency.  DNA Polymerase Taq Polymerase This enzyme is obtained from the bacterium Thermus aquaticus or recombinant Escherichia coli. A characteristic property of this enzyme is its thermostability in the temperature range of 37 to 94°C. Activities of Taq Polymerase: DNA synthesis from the 5' to 3' end (requires a single-stranded template and a DNA or RNA primer). Degradation of single-stranded or double-stranded DNA (5'-3' exonuclease activity). No 3'-5' exonuclease activity (inability to remove incorrectly inserted nucleotides), and the rate of misincorporation is 2x10-4 to 2x10-5.  Taq Polymerase Taq Polymerase Concentration – 1 U per reaction (0,04-0,1 U/μl). Higher concentrations of the polymerase can lead to the synthesis of non- specific products. 1U is described as incorporationod 10nmol od dNTP in a period of 30 min at temp. 70 C If various inhibitors are present in the reaction mixture (e.g., when the DNA template used is not thoroughly purified), higher polymerase concentrations are recommended.  Elongation temperature The elongation temperatures range from 65 to 75°C, with 72°C being the most common. The elongation rate varies depending on the polymerase and can range from 1 kb/min to several kb/min. In most reactions, a rate of 1 kb/min is generally assumed. Lowering the elongation temperature (60 - 68°C) at the expense of extending the duration is beneficial in scenarios such as: - when greater accuracy in the synthesis of a new strand is required, e.g., in cyclic sequencing reactions. - when the amplified fragment will be cloned into expression vectors.  Reaction buffer The PCR reaction buffer is specific to a particular polymerase and is typically supplied along with the polymerase. It stabilizes the pH of the reaction mixture. The buffer contains monovalent potassium chloride (KCl) ions. The standard KCl concentration is approximately 50 mM, allowing for the amplification of fragments larger than 500 base pairs. Higher KCl concentrations (70 - 100 mM) improve the amplification of fragments below 500 base pairs.  Reaction mixture Besides the Tris buffer at pH=8, the reaction mixture often contains additives such as: Bovine Serum Albumin (BSA) Ammonium sulfate [(NH4)2SO4] Triton (a chemical detergent) These additives can stabilize the polymerase and modify interactions between the template and primers.  Risks False positive results Post-amplification product contamination. Contamination of reagents. Contamination by an organism during the preparation of positive controls or positive samples. False negtive results Inhibitors of the reaction. Errors in sample volume. Human error. Poor quality of isolated nucleic acids. Equipment malfunction (thermocycler). Issues related to reagents (magnesium concentration, primer synthesis).  Necessary Control Reactions Positive control In a PCR reaction containing all the components, special DNA template is added known to contain a primer-binding sequence. It allows for its amplification, resulting in a PCR product. The absence of the expected product in such a reaction indicates incorrect preparation (addition) of one of the components. It may also indicate the use of an incorrect PCR program (incorrect temperature, cycle number, etc.). The presence of DNA product after the reaction indicates that the PCR reaction was prepared and executed correctly.  Necessary Control Reactions Kontrola negatywna In a PCR reaction containing all the components, water is added instead of a DNA template. In this reaction, no product should be generated, confirming that the reaction components were not accidentally contaminated with DNA template. The presence of a product after the reaction indicates contamination of the reaction components with undesired DNA template. The results of the remaining tested samples cannot be properly interpreted.  Varieties of PCR END POINT PCR Most simple PCR, the amplified DNA product is analized mainly qualitatively by gel elecrophoresis. MULTIPLEX PCR Multiplex PCR is the simultaneous amplification of multiple genomic regions in a single tube, using different pairs of primers specific to the sequences under investigation. REAL TIME PCR (quantitative PCR) It uses fluorescence as parameter do detect and measure quantity of amplified DNA after every PCR cycle during ongoing PCR reaction No gel electrophoresis needed for analysis. End point PCR vs. Real Time PCR The analysis of the PCR Analysis of the product during product after the PCR reaction the PCR reaction (observing DNA copy number changes) Qualitative analysis Quantitative analysis (quantitative PCR, qPCR) Further product preparation, Determining the initial relative genetic engineering or total amount of genetic material in the tested sample  Real Time PCR (PCR in real-time) Fluorescent dye SYBR Green not bound to DNA (no light emitting) Fluorescent dye SYBR Green bound to DNA (light emitting - measured by a detector) Foto detektor  Reverse Transcriptase PCR Study and analysis of mRNA material. Before PCR, isolated high-quality mRNA material needs to be „converted” into single-stranded cDNA. For this purpose, reverse transcriptase (also known as reverse transcriptase or RNA-dependent DNA polymerase) is used, which is an enzyme that synthesizes a DNA strand based on an RNA template. Reverse transcriptase is an enzyme used by all retroviruses and retrotransposons, and it "transcribes" genetic information from the virus or retrotransposon's RNA into DNA. The first DNA strand produced from mRNA is called cDNA and is synthesized from a reverse primer or an oligo dT sequence TTTTTTTTTTT (complementary to the poly-A tail of the mRNA molecule).  Reverse Transcriptase PCR (RT-PCR) cDNA generated through the process of reverse transcription is much more stable than mRNA, but only one cDNA molecule is produced from each mRNA molecule. Therefore, cDNA needs to be amplified using a quantitative real-time PCR method, i.e., qPCR. Results obtained in this manner indicate the quantity of mRNA in the tested sample, which translates, for example, into the gene expression level of the specific RNA. Quantitative real-time PCR, often referred to as qPCR, combined with the preceding reverse transcription reaction, is a common research and diagnostic method (e.g., for SARS-CoV-2) because the result is known at the end of the reaction.  https://www.youtube.com/watch?v=2KoLnIwoZKU DNA Electrophoresis  Electrophoresis Electrophoresis is an analytical technique employed to separate molecules with different masses what allows for the assessment of their sizes. Electrophoresis involves the movement of electrically charged particles, dissolved or suspended in an electrolyte solution, under the influence of applied voltage. The ability of ions and macromolecules to move in their surrounding environment depends on: The size of the ion and the electrical charge accumulated on it. The properties of the electrolyte, such as its ionic strength and pH value.  DNA and Protein Electrophoresis DNA and proteins undergo electrophoresis because they carry a charge. At neutral pH, DNA has a negative charge and migrates towards the positive anode. In the case of proteins and peptides, their electrical charge depends on environmental conditions. Effective electrophoretic separation depends on maintaining a constant pH value of the electrophoretic buffer. The electrophoretic buffer must be an electrolyte.  Electrophoretic Carrier Electrophoresis can be conducted in solutions, but it is characterized by low resolution. Electrophoresis with electrophoretic carriers finds much wider applications. They provide greater: stability, repeatability, resolution The carrier should not react with the protein or DNA (separation based on physical rather than chemical properties). Changing the carrier concentrations should allow for the adjustment of pore size, and consequently, the selection of the range of molecule separation  Electrophoretic Carrier Electrophoretic carrier (e.g., filter paper, cellulose nitrate, agarose, polyacrylamide, and others): Stabilizes the electrolyte, Contributes to better separation of macromolecules The use of porous carriers enhances the separation effect by additionally fractionating macromolecules on the basis of molecular sieving  Electrophoretic Carrier Standard electrophoresis Gel electrophoresis DNA loaded into a buffer gel well cut in the gel buffer electrophoresis electrophoresis DNA migrates toward the anode, but separation DNA separates into bands with by size classes occurs to a limited extent fragments of different sizes smallest  Agarose gels Agarose is a natural polysaccharide, a polymer derived from galactose. It is obtained from edible agar (a type of red seaweed). Agarose is readily soluble in water and forms a gel reversibly at room temperature. The pore sizes of agarose can be adjusted by using different concentrations (higher concentrations result in denser and smaller pores). Typically, gels with agarose concentrations of 0.4 - 4.0% are prepared and used in horizontal electrophoresis apparatuses..  Agarose gel electrophoresis Advantages of agarose gel electrophoresis: A wide range of separated DNA fragments, - from several base pairs to 40,000 base pairs (in a constant electric field), - in the range of millions of base pairs (in a variable electric field), Easy gel preparation, Non-toxic Disadvantages of agarose gel electrophoresis: Low mechanical strength of agarose gels, Limited resolving power of agarose gels, Agarose Gel Electrophoresis  Polyacrylamide Gels These gels are prepared from a solution of acrylamide monomers and cross- linking substances. The most commonly used cross-linking substance is N,N'-methylenebisacrylamide (bisacrylamide). The polymerization reaction, essentially a free radical polymerization reaction, can be initiated chemically or photochemically. The degree of cross-linking and pore sizes can be regulated by adjusting the concentrations of acrylamide and bisacrylamide.  Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis enables: separation of protein molecules with molecular weights ranging from 5 kDa to 300 kDa, separation of polynucleotides ranging in size from 5 to 2,000 base pairs, Note! Acrylamide in its monomeric form is a highly potent neurotoxin.  Electrophoresis Chambers Depending on the way the electrophoretic carrier is positioned, we distinguish: vertical electrophoresis (for proteins), horizontal electrophoresis (for DNA), capillary electrophoresis (for DNA)  Vertical Electrophoresis A - tube elektrophoresis B - plate electrophoresis plates electrode gel tube buffer gel  Horizontal Electrophoresis A - submarine electrophoresis (agarose gel). B - semi-dry electrophoresis (polyacrylamide gel). gel electrode buffer gel  Zone Electrophoresis The basic and most commonly used type of electrophoresis. It takes place in a carrier (under native or denaturing conditions) in which the electrolyte maintains a constant pH throughout its volume. The difference in migration distances of individual macromolecules directly arises from the difference in their electrophoretic mobility in the carrier in the presence of an electric field.  DNA migration in agarose gel The migration rate of linear dsDNA molecules is inversely proportional to the number of base pairs. Specifically, it is inversely proportional to the log10 of the molecular weight. Therefore, the migration rate depends on the size/mass of the molecule rather than its charge. The conformation of DNA - supercoiled circular form (CCC), open circular (OC), and linear (LIN) DNA forms migrate at different rates in the gel. The relative mobility of these forms depends on: agarose concentration, OC form current intensity, ionic strength of the buffer, density of CCC supercoils LIN form CCC form  DNA Electrophoresis Process The sample DNA solution is mixed with a loading buffer and a dye that also migrates in the electric field, allowing the observation of the electrophoresis front.  Staining Completion of electrophoretic separation or transfer does not end the separation procedure. Most proteins and nucleic acids are not visible in white light. Staining (visualization) of them in gels or on membranes is necessary. This is required to obtain information about the distance of their migration and their quantity in the band or spot (the region containing the protein in a 2D separation). This process allows for both qualitative and quantitative analysis of the separated macromolecules.  Dyes Coomassie Brilliant Blue or Amido Black: The dye is added to the solution that fixes the position of the protein in the gel, and then the excess dye is washed away. Only the dye bound to the proteins remains. This method allows the detection of 1 μg of protein in a band. Silver Staining: This method allows the detection of 10 ng of protein in a band. It can also be used to stain nucleic acids and oligonucleotides. The sensitivity of detection is similar, around 10 ng of DNA per band.  Dyes Fluorescent Dyes: Traditionally, oligonucleotides are stained using ethidium bromide (fluorescent properties). The separation image is visualized under UV light (approximately 300 nm). Ethidium bromide is carcinogenic! Alternative dyes with minimal toxicity are available, such as Midori Green. The sensitivity of detection is comparable to or slightly better than silver staining. Radioisotope Staining: This method offers very high sensitivity but requires increased safety measures  Staining Midori Green Ethidium Bromide  Documentation of electrophoretic separations The documentation of separations performed in polyacrylamide gels involves their drying. This is achieved using filter paper and/or cellophane. Dried gels can be stored indefinitely. However, agarose gels can not be dried. The simplest way to preserve the information contained in an agarose gel is to take a photograph of it. Gel Electrophoresis Simulation 1% 50min 1% 100min 3% 50min 3% 100min