عرض تقديمي في PowerPoint

أمنية عبدالوهاب أحمد محمد. 101703525 أحمد عادل محمد العطار. 101701998 مصطفي عمر عبدالعزيز احمد. 101702968 عمر عماد الهنداوى. 101701099 عمر ماهر عبدالعظيم محمد. 101700475 مؤمن سيد محمد خير. 101701493 عمر علي عبدالفتاح نجم. 101700812 محمد سمير عميرة. 101703493 محمد حمدي علي العكاز ى . 101703133 محمد محمود صلاح حسين. 101701777.

Real-time PCR What is Real-Time PCR? “A technique used to quantify the nucleic acid (DNA/RNA) present in a sample, during the PCR reaction is known as a real-time PCR or quantitative (q)PCR ” In other words, “A molecular biology technique used to monitor the amplification of the target DNA/RNA sequence is referred to as real-time PCR or quantitative PCR.” The principle of real-time PCR: The principle of real-time PCR relies on the use of fluorescent dye . “ The amount of the nucleic acid present into the sample is quantified using the fluorescent dye or using the fluorescent labeledoligos.” When a dye or probe binds with the target template, it releases a fluorochrome which resultantly emits fluorescence for the detector to detect. The detector captures a signal as a positive template amplification.

Two types of chemistry are available for the real-time quantitative PCR: DNA binding dye (Intercalating dye-based method) Sequence-specific probe (Hydrolysis Probe-based detection method).

DNA binding dye: T he DNA binding dye method is the best technique for real-time detection. The dye has its own fluorescence. Once the dye binds to the double-stranded DNA the fluorescence emitted by the dye increases 100 to 1000 fold than the original signal. However, the original dye fluorescence is taken as the baseline (as a reference) for the detection. The method is rapid, quick, reliable and cost-effective. Also, the chance of error in the experiments is less and the reaction setup is simple & easy to use. The result of the experiment depends on the specificity of the primers used in the PCR reaction. Because even though the primers remain bound non-specifically, the DNA binding dye binds to the non-specific sequence and gives the fluorescent signals. As the dye detects the double-stranded DNA to bind, even if the dsDNA is non-specific, the dye binds to it..

Therefore the chance of the non-specific detection is high in the SYBR green dye-based method. The SYBR green is one of the most popular dyes used in real-time PCR. The sensitivity of the experiment is limited. It is suitable for the determination of sensitive templates A melting curve analysis helps to identify non-specific bindings during the reaction. Melting curve analysis : After completion of the amplification reaction and capturing fluorescence signals, melting the template (again) determines non-specific bindings if any. During melting, at a high temperature, the template starts denaturing which consequence dye dissociation and reduce fluorescence. Varied heat transition reported shows the amount of non-specific products while the gradual decrease in fluorescence shows the presence of specific amplification product. A larger sequence need more time and higher temperature for melting while non-specific amplicons needs lower and varied temperature to melt so gives more shorter curves in a graph. The fluorescence vs melting temperature graph is also called a dissociation curve and the method is called a dissociation curve analysis . Two common dyes employed in the real-time PCR are the SYBR green and EvaGreen, notably, the technique is used in the validation of other assays such as DNA microarray..

. Probe-based detection method: The method used the single short sequence-specific probes which are of three types: A linear probe ,molecular beacons and scorpion probe . Linear probe: Linear probes are the TaqMan probe, which relies on the activity of Taq DNA polymerase. The probes structurally consist of labeled short single-stranded sequence-specific DNA molecules that are radio or fluorescent-labeled. Here the probe is labeled with the fluorescent dye described as a reporter molecule, situated at the 3 ’ end. The other 5 ’ end has the quencher dye which is in close proximity to the reporter dye and quenches the fluorescence of the reporter dye. Now , in the probe base method, not only the probe but the Taq DNA polymerase plays an important role. The Taq DNA polymerase used in the real-time PCR has the 5’ to 3’ exonuclease activity, which removes the probe by extending the DNA. Once the probe dissociates the reporter molecules emitted fluorescent light. Because, if the DNA (the sequence of our interest) is amplified, the reporter molecule is unquenched and releases the fluorescence The amount of fluorescence released during each run is directly proportional to the amount of DNA amplified during the reaction ..

Steps and procedure of real-time PCR: The quantification is achieved by amplifying and monitoring the DNA or RNA present in the sample. For the quantification of the gene expression, the RNA is quantified into the real-time PCR. If DNA is present in the sample in a higher quantity, amplification and quantification start at the early stage of the reaction; otherwise, the amplification starts in the late stage. As like the conventional PCR, there are three main steps in real-time PCR; Denaturation Annealing Extension Denaturation occurs at 94°C where the double-stranded DNA is denatured and two single-stranded DNA is generated. The DNA is melted. This single-stranded DNA is the sight of the annealing for the primers in the later step of the amplification. Annealing occurs at 55°C to 66°C in which the sequence-specific primer bind to the single-stranded DNA. Along with it, the fluorescent dye or the probe bind to the DNA sequence too. Extension occurs at 72°C at which the Taq DNA polymerase activated highest. In this step, the Taq adds dNTPs to the growing DNA strand. Note: if the amplicons are less, combine the extension step with the annealing step (for real-time PCR only)..

Components used into the real-time PCR: Similar to conventional PCR, the real-time PCR reaction contains almost the same components except for the fluorescent dye or fluorescent-labeled probe. Let’s start with the dNTPs dNTPs are added during the synthesis of the growing DNA strand by the Taq DNA polymerase. The dNTPs remain the same as the conventional PCR. Taq DNA polymerase: Normal Taq cannot work efficiently for the real-time PCR, instead always use the hot-start Taq DNA polymerase. The hot start Taq DNA polymerase is the best choice for the quantification. MgCl2: Magnesium ion also plays a crucial role in the amplification during real-time PCR. However, the concentration of the Mg2+ ions is different from the conventional PCR . Use 3 to 5mM of MgCl2 in the real-time PCR..

Template: 100pg to 1microgram template DNA is sufficient for real-time PCR. We required only 100 copies of genomic DNA/RNA fragments for the amplification and to start the reaction. The template DNA or RNA must be pure and free from any contaminants. Primers: The primer should be shorter, it can amplify only 100 to 160bp fragments, and avoid longer amplicons. It must be unique and contains 50% GC at the end of the 3’ end. It should be 18 to 20 nucleotides long. The primer contains all these criteria that are the best for a real-time PCR assay The procedure of the real-time PCR starts with the extraction. The DNA or the RNA is extracted and quantified using the ready-to-use kits. Set the cyclic condition of the PCR and put the samples inside the machine. After the amplification, standard curve analysis or relative quantification is performed, instead of agarose gel electrophoresis. For quantification of different types of samples, Endpoint PCR or real-time PCR can be performed..

Advantages of Real-time PCR: The method is cost-effective . The conventional PCR method is more costly than the qPCR due to the use of so many other chemicals and agarose gel electrophoresis. It is time-efficient . Definitely, it is. The average time consumed by the PCR reaction along with the agarose gel electrophoresis and data interpretation is approximately 4 to 4.5 hours. Contrary, real-time qPCR gives results in ultra-fast time. The average duration of the qPCR reaction is around 30 minutes to 2 hours. More sensitivity and specificity . The quantitative real-time PCR method is more sensitive, specific and efficient. Though the probes and primers are highly sequence-specific, if any non-specific bindings occurred, it is monitored immediately during the reaction. Also, the main reaction or the quantification of our template cannot be influenced by the non-specific bindings..

Fewer templates required: The overall assay required less amount of the template material. It required 1000 folds less template DNA or RNA for the reaction to occur as compared with the conventional PCR. Melting curve analysis: The main advantage of quantitative PCR is the confirmation of the analytes through the melting curve analysis. We can measure and quantify how many amplicons are generated and how many non-specific or primer-dimers are formed during the PCR reaction by doing the melting curve analysis . The major advantage over the other PCR technique is the quantification. It quantifies the template DNA or RNA present in the sample . Only Real-time is sufficient: No post PCR processing and data processing is required in the quantitative real-time PCR. As with conventional PCR, agarose gel electrophoresis gel-based interpretation is not needed in the qPCR..

Limitation of real-time qPCR(real time PCR): Although the advantages of the quantitative RT_PCR are far more than the conventional PCR, still the technology has several limitations. The instrument itself is too costly as compared with conventional PCR The multiplexing is still limited in Real-time PCR. Kits are not available for all kinds of genes and disorders. The technical and standardized protocols are limited. Higher expertise and technical skills are required for developing a novel qPCR assay.

Applications of quantitative PCR: From gene quantification to gene expression, the real-time qPCR is the ocean of different applications in different fields. Disease diagnosis: One of the most powerful uses of any PCR technique is in the inherited disease diagnosis. The real-time PCR is used in the diagnosis of a single gene and multigenic disease. It is used to quantify the mutated gene in the disease patient. The quantitative real-time PCR is even used in the determination of copy number variation in different tissues for different inherited disorde microRNA analysis: MicroRNAs are smaller RNA molecules of 20 to 25 nucleotides in length. It plays an important role in the gene regulation pathway. The qPCR assay is used to quantify the microRNA from different tissues. By quantifying it we can estimate the gene expression level in different tissues influenced by the microRNA. Cancer detection: Circulating tumor cells contain the mutant mRNA which transports to different tissues if it is malignant. By quantifying the total mRNA against the mutant mRNA into the samples of cancer or from the cancer cell. The stage of cancer can be determined by which the severity of the carcinoma can be estimated. The mutant mRNA is the best biomarker for gene expression studies for cancer. Furthermore, real-time quantification can be helpful in measuring the recovery in cancer therapy. After each therapy, the gene expression level of the mutant oncogenic cancer genes from the affected tissues is determined. By doing RT-PCR, the success of the therapy can be estimated. Therefore, cancer diagnosis, prognosis and monitoring to the response of therapy can be done using the real-time qPCR..

Microbial load testing: The accurate microbial load testing from any biological sample is nearly impossible without the qPCR. Microbial load in the fermented sample, soil sample, water sample, food and food spoilage can be accurately estimated by the real-time PCR. Also, the estimation of the active microbial load is determined. Additionally, microbial risk assessment can also be possible by this method. GMOs detection: Genetically modified organisms are the organism whose genetic makeup is altered using the genetic engineering or transgenic technique specifically into plant, animal and microorganism. Now, this is something interesting, Inserting DNA through the vector is not sufficient to do . So for the successful development of GMOs, one has to estimate the expression or the protein formed by the inserted gene. For that, only gene detection cannot help, the mRNA expression quantification through real-time PCR helps to detect the amount of the gene expressed into the GMO. Further, the amount of inheritance of that inserted gene is determined as well..

Genotyping and quantification of pathogens: Microbial infections are the second most common reason for worldwide mortality and morbidity. Unlike conventional PCR (which can only detect a single or a few strains and genotyping), RT-PCR can quantify the amount of infection and measure microbes present in a sample. Melting curve analysis, dissociation curve analysis and Ct value analysis help in investigating the severity of the infection. The most recent examples are the detection and quantification of COVID-19 coronavirus and H1N1 swine flu. q(RT)- PCR helped in both types of pandemics which quantify the absolute amount of virus present in a sample in rapid time. The qPCR is applicable in the Identification, characterization, genotyping and quantification of an infectious pathogen..

RT_ PCR is also used in forensic studies evolutionary studies mutation creation fossil studies in other applied fields.