PCR Multiplex PCR PDF

PCR & MULTIPLEX PCR POLYMERASE CHAIN REACTION SSCB 3713 - TECHNIQUES IN MOLECULAR BIOLOGY GROUP 1 Polymerase Chain Reaction  What is PCR? Polymerase Chain Reaction (PCR) is a technique used to make millions of copies of a specific DNA segment. By simulating DNA replication in a lab, PCR allows sc...

PCR & MULTIPLEX PCR POLYMERASE CHAIN REACTION SSCB 3713 - TECHNIQUES IN MOLECULAR BIOLOGY GROUP 1 Polymerase Chain Reaction  What is PCR? Polymerase Chain Reaction (PCR) is a technique used to make millions of copies of a specific DNA segment. By simulating DNA replication in a lab, PCR allows scientists to amplify tiny amounts of DNA, making it easier to study or analyze. The process involves three main steps: denaturation , annealing , and extension. This cycle repeats, doubling the DNA each time, until a large amount of the target DNA is generated.  PCR COMPONENTS Buffer plate DNA Primers set Tem A polymeras dNTPs DN e PCR Principle 1. Denaturation: The DNA sample is heated to around 94-98°C, causing the double-stranded DNA to separate into two single strands. 2. Annealing: The temperature is lowered (typically 50-65°C) to allow short DNA primers to attach to complementary sequences on each DNA strand. These primers mark the starting points for DNA synthesis. 3. Extension (Elongation): The temperature is raised to 72°C, optimal for DNA polymerase to add nucleotides to the primer and synthesize new DNA strands. BASED ON THIS ARTICLE Roles of primer & DNA polymerase in PCR PCR PRIMERS REGION TO BE COPIED Short pieces single-stranded DNA (~20 5' 3' nucleotides in length) TEMPLATE DNA 3' 5' Two primers are used in each PCR reaction, each of them will bind to target 5' 3' region by complementary base pairing Provide as starting point for DNA PRIMER 1 5' 3' 3' 5' PRIMER 2 synthesis 3' 5' Determine the PCR specificity by binding to specific sequences of DNA.  CHLOROPLAST GENOME MAP Fagopyrum dibotrys chloroplast genome 159,919 bp  Ladder rbcL ITS1 ITS2 1000 900 800 700 600 500 400 300 “The DNA barcode primers, rbcL, ITS1 and ITS2, produced amplicons of 599 bp, 300–400 bp and 300– 200 500 bp, respectively. In analyzing the sequences, rbcL was found to exhibit the most extensive sequence 100 length (502–509 bp), followed by ITS1 (219–327 bp) and ITS2 (241–250 bp).”  Roles of primer & DNA polymerase in PCR DNA POLYMERASE 3' 5' Enzyme that synthesizes new DNA TEMPLATE DNA 5' 3' strands complementary to template strand. Most commonly used in PCR: Taq 3' 5' PRIMERS BIND TO 5' 3' polymerase from Thermus 3' 5' THE TEMPLATE 5' 3' aquaticus Heat stable & most active around 70°C 3' 5' DNA polymerase will add TAQ POLYMERASE EXTENDS THE 5' 3' nucleotides to the 3' end of the 3' 5' PRIMERS 5' 3' annealed primer, extending the DNA strand. DATA OUTPUT ANALYSIS Almost to no bands were identified, possibly due to: Insufficient DNA template: The template DNA might be a bit too diluted or degraded. Suboptimal cycling condition: The annealing temperature might be too high, preventing primer binding to the template. Primer issues: Poorly designed primers that do not bind properly to the template. Introduction to Multiplex PCR What is Multiplex PCR? Multiplex Polymerase Chain Reaction (PCR) An advanced variation of the conventional PCR method that enables simultaneous amplification of multiple target DNA sequences inside a single reaction.  Characteristic of primer design PRIMER LENGTH & MELTING TEMPERATURE Length : typically 18 - 22 nucleotide long Tm : similar Tm for all primer in multiplex reaction PRIMER SPECIFICITY Ensure each primer is specific to its target sequence and does not bind to other non- target sequence & avoid self-complimentary and Hairpin formation AMPLICON SIZE The size of the amplified product should be significantly different to allow easy separation and detection. ( 100 - 500 bp ) PRIMER CONDITION It's often necessary to optimize primer concentrations for specific reactions. (0.2-0.5 µM for each primer)  PRIMER DESIGN SOFTWARE 1. Primer3 Primer3 is a fundamental tool for designing individual primer pairs. It analyzes a given DNA sequence and outputs potential primer pairs based on parameters like length, GC content, and melting temperature. Primer3 Input. (n.d.). https://primer3.ut.ee/  PRIMER DESIGN SOFTWARE 2. PrimalScheme PrimalScheme is a more advanced tool specifically designed for multiplex PCR. It leverages Primer3 to generate candidate primer pairs, but it also incorporates additional features to optimize primer design for multiplex reactions. Source : Overview of multiplex primer design using PrimalScheme online primer design tool Source IMAGE A show input need to enter target sequences for Zika and Chikungunya along with desired parameters like primer length, Tm, product size. IMAGE B Primer table displaying designed primer pairs for each viruses IMAGE C Pool 1 & 2 refer to diff. sets of primer designed to amplify specific region of the target genome. Coloured Boxes represent specific region of the desired sequences that will be amplified by each primer pair. ( generates by PrimalScheme )  Principles of Multiplex DETECTION METHODS GEL CAPILLARY REAL-TIME PCR ELECTROPHORESIS ELECTROPHORESIS Simple & cost effective Highly sensitive & Precise method for separating method to visualise and quantitative method allows and quantifiying DNA fragments. separate amplified product for the detection of amplified ( used for genotyping and based on size product in real time. mutation analysis ) Application of Multiplex PCR ogen Detec BASED ON PAPER netic Testin th tio G e g Pa n analysis result how multiplex is applied? rensic Scienc Fo e BASED ON THIS ARTICLE As of September 20, 2020, Malaysia reported 10,219 confirmed COVID-19 cases, with approximately 33% linked to a Tablighi Jamaat religious mass gathering that took place in Kuala Lumpur from February 27 to March 3, 2020. This event significantly contributed to community transmission during Malaysia's second wave of the pandemic  HOW Sample Collection MULTIPLEX Viral RNA Extraction Two pools of 109 nCoV- PCR cDNA Synthesis 2019/V3 primer sets were used to target multiple regions of the SARS-CoV-2 IS Multiplex PCR Setup genome in a single PCR reaction. Q5 High-Fidelity DNA APPLIED? Amplification of Polymerase was employed for the amplification Overlapping Amplicons process. Pooling & Purification Library Preparation & Sequencing  ANALYSIS RESULT Identification of Identification of Lineage B.6 Mutation The study also detected The study identified the B.6 various mutations, including lineage as the predominant a significant nsp3-C6310A strain responsible for substitution that caused community transmission in reduced sensitivity in a Malaysia commercial diagnostic real- time PCR assay. BASED ON THIS ARTICLE What are the Quality Control measures applied in this paper to ensure the reliability of sequencing results? ✅ How to control the error rate associated with Taq polymerase during PCR amplification 1 - POSITIVE CONTROL Positive control of The Zika virus reference strain PF13/251013-18 is used as it has viral copy numbers similar to those of the clinical samples to ensure that the protocol is generating expected results. 2 - NEGATIVE CONTROL Negative sequencing controls should be handled like clinical samples, not just with water controls. For example, if swabs are used for sample collection, an unused swab should go through RNA extraction and PCR as a negative control. 3 - SEQUENCING NEGATIVE CONTROL Negative controls should be sequenced even if no DNA is detected. This helps identify any contaminating DNA that might also appear in other samples. Comparing the number of reads in negative controls with positive samples can show the level of contamination.  BASED ON THIS ARTICLE What are the Quality Control measures applied in this paper to ensure the reliability of sequencing results? ✅ How to control the error rate associated with Taq polymerase during PCR amplification ✅ How error rate associated with Taq Polymerase can happen in multiplex PCR? In multiplex PCR, the use of Taq polymerase can lead to an increased error rate, primarily due to its intrinsic limitations and the complexities of amplifying multiple targets simultaneously. How much generally Taq polymerase relative error rate? Typically around 1 in 10,000 base pairs, due to its lack of proofreading ability. This means that it does not correct errors during DNA synthesis, which can lead to mutations in the amplified DNA. How does the tax polymerase error rate is controlled in this paper? 1. Optimization of PCR Conditions : The paper notes that PCR conditions require optimization, particularly when using primers generated by software to enhance the specificity and efficiency of the amplification process. 2. Sequencing Controls : The inclusion of positive and negative controls in sequencing runs allows for the assessment of amplification fidelity.  Commercial Example of Taq Polymerase with Proofreading ability Q5 Hot Start High- Fidelity DNA Polymerase ✅ Pfu DNA polymerase Platinum SuperFi DNA Polymerase Phusion DNA polymerase Example of Kit for Multiplex PCR  Qiagen Multiplex PCR Kit For 100 x 50 µl multiplex PCR reactions: 2x QIAGEN Multiplex PCR Master Mix Qiagen Multiplex PCR Kit (providing a final concentration of 3 mM MgCl2, 3 x 0.85 ml), 5x Q-Solution (1 x 2.0 ml), RNase-Free Water (2 x 1.7 ml) ADVANTAGES : High Specificity Reduced Optimisation Requirements Increase Efficiency STEPS IN USING : 1. Prepare Reaction Mixture by gathering necessary components. 2. Set up Thermal Cycler 3. Run PCR 4. Analyse data by using Gel Electrophoresis.  P SUGAR Comparison PCR and Multiplex PCR TARGET AMPLIFICATION : PCR : Single target sequence MULTIPLEX PCR : Multiple target sequences simultaneously PRIMER PAIRS : PCR : One pair of reverse and forward primers MULTIPLEX PCR : Multiple pair of reverse and forward primers EFFICIENCY PCR : Less efficient, as each target requires a separate reaction MULTIPLEX PCR : More efficient, as multiple targets are amplified in a single reaction REACTION COMPLEXITY : PCR : Simpler reaction MULTIPLEX PCR : Complex reaction for effectivity APPLICATION : PCR : DNA cloning, sequencing, genotyping, forensic analysis MULTIPLEX PCR : Genetic testing, pathogen detection, food safety testing, environmental monitoring QUIZ TIME !!!!  Thanks! Do you have any questions?