DNA Amplification by PCR PDF

DNA AMPLIFICATION BY Polymerase Chain Reaction (PCR) Starting with double-stranded DNA molecule, process involves number of amplification cycles. PCR requires three step amplification cycle. Dr Maimunah Mustakim preencoded...

DNA AMPLIFICATION BY Polymerase Chain Reaction (PCR) Starting with double-stranded DNA molecule, process involves number of amplification cycles. PCR requires three step amplification cycle. Dr Maimunah Mustakim preencoded.png  LEARNING OBJECTIVES AT THE END OF THE LESSON, STUDENTS ARE ABLE TO EXPLAIN/DESCRIBE/DISCUSS: I. Principles of gene amplification by PCR. II. PCR components and optimization technique. preencoded.png PCR Amplification Cycle Denaturation 1 Double-stranded DNA denatured by heat. 2 Annealing Primers anneal to complementary sequence of target DNA. Extension 3 DNA synthesis occurs with heat-stable DNA polymerase. preencoded.png PCR: Amplifying DNA 1 Exponential Amplification DNA is amplified exponentially. 2 Rapid Replication Creates millions of copies of given region of DNA in matter of hours. 3 Primer Specificity Technique exploits specificity of primers. 4 Selective Replication Allows for selective replication of chosen regions. preencoded.png PCR: Target DNA (GENE) Target DNA Specific gene. Sample Amplification Large amounts of DNA can be produced from very small sample. Contamination Prevention Care must be taken to prevent contamination with external source of target DNA. False-Positive Results Basis for false-positive test results. preencoded.png PRINCIPLES OF PCR AMPLIFICATION Step 1 Each cycle of PCR amplification consists of a defined number of reaction steps. Step 2 The steps are designed using temperature and duration time to denature the template, anneal the two oligonucleotide primers and extend the new complementary DNA strands by polymerization. Step 3 The target DNA is denatured by heating to 95°C or higher for 15–120 seconds. preencoded.png PCR COMPONENTS Buffer Provide suitable environment for optimum activity and stability of the DNA polymerase. MgCl2 Cofactor for the DNA polymerase. dNTPs Building blocks of a new DNA strand. Forward primer and reverse Initiation point for synthesis of a primer new strand. Thermostable DNA polymerase Incorporate new nucleotides which are complementary to template DNA. preencoded.png OPTIMIZATION OF PCR COMPONENTS Magnesium Enzyme Concentration Primer Design Crucial factor affecting the An appropriate amount of enzyme PCR primers - length from 15–30 performance of thermostable in PCR reactions is essential to bases, designed to flank the region enzymes. Reaction components, avoid production of artifacts - can of interest. including template DNA, chelating be seen as smearing of bands in agents present in the sample (e.g., ethidium bromide-stained agarose EDTA or citrate), dNTPs and gels. proteins, can affect the amount of free magnesium. preencoded.png PCR: Primer Design & Annealing Primer Annealing Temperature The sequences of the primers are a major consideration in determining the optimal temperature of the PCR amplification cycles. DNA Template Successful amplification of the region of interest is dependent upon the amount and quality of the template DNA. Using Controls in PCR Successful amplification of the control template will indicate that the genomic DNA sample is compatible with the reaction conditions used. preencoded.png RNA Handling and Amplification for Molecular Biology Research RNA, a crucial molecule in gene expression, is highly susceptible to degradation. Proper handling and preservation are essential for accurate molecular biology experiments. Dr Maimunah Mustakim preencoded.png LEARNING OUTCOMES AT THE END OF THE LESSON, STUDENTS ARE ABLE TO EXPLAIN/DESCRIBE/DISCUSS: The fundamental knowledge and practical skills required for handling and amplifying RNA in a molecular biology research setting. From understanding RNA structure and function to mastering RNA handling techniques, performing RNA extraction, and applying RNA amplification methods, you are now equipped to tackle various research challenges. Remember that ethical considerations and data reproducibility are crucial to scientific integrity, ensuring that your research findings are reliable and contribute to advancing scientific knowledge. preencoded.png RNA Structure and Function RNA, or ribonucleic acid, is a single-stranded nucleic acid molecule that plays a crucial role in protein synthesis and gene regulation. Its structure is similar to DNA, featuring a sugar-phosphate backbone and nitrogenous bases, but instead of thymine, RNA contains uracil. The structure of RNA allows it to fold into complex three-dimensional shapes, creating diverse functional roles. There are various types of RNA, each with distinct functions. For example, messenger RNA (mRNA) carries genetic information from DNA to ribosomes, where protein synthesis occurs. Transfer RNA (tRNA) delivers amino acids to ribosomes during translation, and ribosomal RNA (rRNA) is a component of ribosomes, essential for protein synthesis. 1 Genetic Information 2 Protein Synthesis Catalyst 3 Gene Regulation Carrier RNA molecules can regulate RNA acts as a messenger RNA forms a crucial part of gene expression by influencing molecule, carrying genetic ribosomes, the cellular machinery the transcription or translation of information from DNA to the responsible for protein genes. ribosomes, where protein production. synthesis takes place. preencoded.png RNA Sample Handling 1 Temperature Control 2 RNase Contamination 3 Dedicated Equipment RNA is extremely sensitive to RNases are ubiquitous enzymes Use micropipettes and tips heat and enzymatic degradation. that readily degrade RNA. Use dedicated for RNA work to Store samples at -80°C, ensuring RNase-free water, wear gloves, prevent cross-contamination. rapid freezing to minimize and decontaminate work Autoclave glasswares at 180°C to degradation. surfaces to minimize eliminate RNases. contamination. preencoded.png Mastering RNA Handling Techniques Handling RNA requires meticulous attention to detail to avoid degradation. RNases, enzymes that degrade RNA, are ubiquitous and can contaminate laboratory equipment and reagents. Therefore, utilizing RNase-free reagents, glassware, and equipment is paramount. This includes working in designated RNase-free areas and wearing gloves to minimize contamination. Proper handling ensures that the integrity of RNA is preserved for downstream applications such as gene expression analysis, RNA sequencing, and other molecular biology techniques. Use RNase-Free Dedicated Work Area Proper Handling Reagents Techniques Ensure all reagents, including Utilize a designated RNase-free Wear gloves, use sterile pipettes, water, buffers, and enzymes, are workspace, preferably with and avoid unnecessary handling RNase-free. dedicated equipment, to minimize to prevent contamination and contamination. degradation. preencoded.png RNA Extraction Protocols RNA extraction is the first crucial step in RNA research. Various methods are available for RNA extraction, each with its advantages and disadvantages. Common methods include TRIzol-based extraction, which uses a reagent that disrupts cells and separates RNA from other cellular components, and column- based kits, which utilize silica membranes to bind RNA. Cell Lysis 1 Disrupt cells to release RNA from the cellular components using a suitable lysis buffer. 2 RNA Isolation Separate RNA from DNA, proteins, and other cellular debris using methods like TRIzol extraction or column-based kits. RNA Precipitation 3 Precipitate RNA using isopropanol or ethanol to concentrate and purify the RNA. 4 RNA Quantification Determine the concentration and purity of the extracted RNA using a spectrophotometer. RNA Integrity Assessment 5 Evaluate the quality of extracted RNA using gel electrophoresis to assess its integrity and detect degradation. preencoded.png Amplification of RNA RNA amplification is a crucial technique in molecular biology that allows scientists to amplify specific RNA sequences for further analysis. Reverse transcription polymerase chain reaction (RT-PCR) is a widely used technique that combines reverse transcription with PCR. Reverse transcriptase enzyme converts RNA into complementary DNA (cDNA) before it can be amplified using PCR. RT-PCR qPCR Digital PCR Reverse transcriptase converts Quantitative PCR allows for the Digital PCR partitions the reaction RNA into cDNA, which is then quantification of specific RNA into a large number of individual amplified using PCR. sequences by measuring the reactions, allowing for very sensitive amplification rate in real-time. and precise quantification of RNA targets. preencoded.png Applications of RNA Amplification RNA amplification techniques play a critical role in advancing molecular biology research across various fields. RT-PCR and qPCR find applications in gene expression analysis, viral RNA detection, and transcriptome studies. Gene expression analysis allows scientists to study how gene expression patterns change in response to various stimuli or under different conditions, while viral RNA detection can be used to diagnose viral infections and monitor treatment efficacy. Gene Expression Analysis Quantifying and comparing the expression levels of different genes under various conditions. Viral RNA Detection Diagnosing viral infections and monitoring treatment efficacy. Transcriptome Studies Analyzing the complete set of RNA transcripts present in a cell or organism, providing insights into gene regulation and cellular processes. preencoded.png RNA Amplification Reverse Transcription RNA is single-stranded, requiring reverse transcription into cDNA for PCR amplification. High quality RNA ensures efficient and reproducible reverse transcription. RT-PCR Reverse transcription PCR (RT-PCR) utilizes reverse transcriptase enzymes to convert RNA into cDNA, enabling amplification of specific gene sequences. preencoded.png Reverse Transcriptase Enzymes Enzyme Source Properties Avian Retrovirus High processivity myeloblastosis and stability. virus (AMV) Moloney murine Retrovirus High processivity, leukemia virus lower stability, (MMLV) and requires a primer. preencoded.png RT-PCR Primers Random Primers Bind randomly to various sites on the RNA template. Gene-Specific Primers Target specific sequences within the gene of interest. Oligo-dT Primers Hybridize to the poly-A tail of mRNA molecules. preencoded.png RT-PCR Process 1 Reverse Transcription RNA is converted into cDNA using reverse transcriptase and primers. 2 PCR Amplification cDNA is amplified using PCR, producing multiple copies of the target gene sequence. 3 Confirmation The amplified gene sequence is confirmed by sequencing to ensure accuracy and identify the gene of interest. preencoded.png preencoded.png Factors Influencing RT-PCR Success RNA Quality Primer Design Reaction Conditions Intact RNA is crucial for efficient Specific and well-designed Optimal temperature, enzyme reverse transcription. Degradation primers are essential for accurate concentration, and reagent quality or contamination can hinder the amplification of the target gene. are critical for efficient RT-PCR. process. preencoded.png Applications of RT-PCR Gene Expression Analysis Quantifying the levels of specific genes in different cells or tissues. Pathogen Detection Identifying the presence of viral or bacterial RNA in clinical samples. Genetic Diagnosis Detecting genetic mutations or abnormalities that can contribute to disease. preencoded.png Improvements in PCR-based Diagnostics This presentation explores recent advancements in PCR diagnostics, showcasing how improvements in technology have enhanced sensitivity, accuracy, and efficiency. Dr Maimunah Mustakim preencoded.png  LEARNING OBJECTIVES AT THE END OF THE LESSON, STUDENTS ARE ABLE TO EXPLAIN/DESCRIBE/DISCUSS: I. Methods in advanced PCR-based technologies. II. Quantitative PCR, Multiplex-PCR etc. preencoded.png Introduction 1 Revolution in Diagnostics PCR technology has revolutionized diagnostics, enabling the detection and analysis of specific DNA or RNA sequences with unparalleled sensitivity and accuracy. 2 Enhanced Applications Recent developments have led to faster, more precise, and adaptable PCR- based diagnostics, expanding their applications in healthcare and research. 3 From Research to Clinics PCR has transitioned from a laboratory tool to a powerful diagnostic tool, allowing for rapid and accurate disease detection and monitoring. preencoded.png High-Fidelity Polymerases Reduced Error Rates High-fidelity polymerases minimize errors during DNA amplification, crucial for applications where accuracy is paramount, such as detecting genetic mutations or conducting sequencing analysis. Enhanced Accuracy These enzymes possess advanced proofreading capabilities, ensuring precise DNA replication, which is essential for reliable results in clinical diagnostics. Reliable Results High-fidelity polymerases are vital for accurate and reliable clinical diagnostics, leading to more accurate disease diagnosis and treatment decisions. preencoded.png https://assets.thermofisher.com/TFS-Assets/LSG/product-images/F534L- 650x600.jpg-650.jpg preencoded.png Hot-Start PCR Technology 1 Minimizing Non-Specific Amplification Hot-start PCR inhibits polymerase activity at lower temperatures, preventing non-specific amplification and improving diagnostic accuracy. 2 Preventing Contamination This technology reduces false positives by minimizing the formation of undesired products, crucial for reliable clinical diagnostics. 3 Enhanced Specificity Hot-start PCR enhances the specificity of PCR reactions, ensuring that only the desired target sequences are amplified, leading to more precise results. preencoded.png  DNA polymerases, Phusion® hot start II high-fidelity PCR master mix Supplier: Thermo Fisher Scientific Phusion® high-fidelity DNA polymerases set a gold standard for high-performance PCR. Reaction set up at room temperature No non-specific amplification and primer degradation during reaction set up Zero-time reactivation due to unique hot start technology https://uk.vwr.com/stibo/low_res/std.lang.al High fidelity (52X Taq) l/43/64/18534364.jpg Fast PCR due to short extension times (15 to 30 s/kb) Robust reactions, minimal optimization needed Increased product yields with minimal enzyme amounts Store at –20 °C preencoded.png Real-Time PCR (qPCR) for Rapid Diagnostics Real-Time Monitoring qPCR allows for real-time monitoring of DNA amplification, providing faster results than traditional PCR, making it ideal for rapid diagnostics. Quantitative Analysis qPCR provides quantitative data on the concentration of target sequences, which is valuable for monitoring disease progression and understanding disease severity. Rapid and Accurate qPCR is widely used for pathogen detection, particularly in infectious diseases like COVID-19, enabling timely diagnosis and treatment. preencoded.png Multiplex PCR Amplifying Multiple Targets Diagnostic Versatility Streamlined Analysis Multiplex PCR amplifies multiple targets This technology is particularly useful for Multiplex PCR simplifies diagnostic simultaneously in a single reaction, detecting multiple pathogens or genes, workflows, providing comprehensive increasing efficiency and reducing enabling clinicians to diagnose various information with minimal sample sample usage. conditions simultaneously. requirements. preencoded.png https://frontlinegenomics.com/wp-content/uploads/2021/07/Picture-4.png preencoded.png PCR-Based Point-of-Care Diagnostics Decentralized Diagnostics Portable PCR platforms bring molecular diagnostics to point-of-care settings, making it accessible in remote areas or resource-limited environments. Rapid Results Point-of-care PCR allows for faster diagnosis and treatment initiation, leading to improved patient outcomes and reduced healthcare costs. Enhanced Accessibility These technologies make molecular diagnostics more readily available, reducing the need for centralized lab facilities and enabling quicker decision-making. preencoded.png  BioFire FilmArray 2.0 Multiplex PCR System The FilmArray® GI Control Panel M238 is intended https://cdn.shopify.com/s/files/1/0397/ for in vitro use as a quality control to monitor the 7153/7572/products/IMG_1264_1300x detection and identification of multiple.jpg?v=1611202389 gastrointestinal pathogens as performed by the FilmArray® Gastrointestinal (GI) Panel assay on the FilmArray® instrument. Detection of the viruses, bacteria and protozoa listed in Table 1 is an important aid to the diagnosis of gastrointestinal illness. https://arcscientific.com/wp- content/uploads/2023/06/image00 0000-2-768x1024.jpeg https://th.bing.com/th/id/R.82432dd9f8e84d9f3c305ab6defc30a3?rik=bR 9AfnA0Aqr2Og&riu=http%3a%2f%2fwww.biomerieux.fr%2fsites%2fsubsi diary_fr%2ffiles%2ffonctionnement-filmarray-biofire- 2.jpg&ehk=x362Zt2Spes%2fYFEC%2fmeMqOWtiAKIVjhqfAUISyVSKoI%3d &risl=&pid=ImgRaw&r=0 preencoded.png Conclusion High-fidelity polymerases Enhanced accuracy, reduced errors in DNA amplification Hot-start PCR Minimized non-specific amplification, increased specificity and accuracy qPCR Real-time monitoring of DNA amplification, rapid and quantitative results Multiplex PCR Simultaneous amplification of multiple targets, increased efficiency and versatility Point-of-care PCR Decentralized diagnostics, faster and more accessible testing preencoded.png Future Directions Ongoing research continues to refine PCR technology, developing new and more powerful applications for diagnostics, personalized medicine, and genetic research. preencoded.png Real-Time PCR: Revolutionizing Molecular Analysis Real-time PCR, or qPCR, is a powerful technique that allows for the detection and quantification of DNA or RNA in real time. This technology has revolutionized molecular biology, enabling researchers to study gene expression, detect pathogens, and perform genetic diagnostics. Dr Maimunah Mustakim preencoded.png  LEARNING OBJECTIVES AT THE END OF THE LESSON, STUDENTS ARE ABLE TO EXPLAIN/DESCRIBE/DISCUSS: I. The principles of q(Real-time) PCR. II. Differentiation of q(Real-time) PCR chemistries. III. Characteristics of qPCR assay. preencoded.png Real-Time PCR: Principle of Operation 1 Fluorescent Detection 2 Direct Measurement qPCR detects PCR products by measuring the generation of qPCR allows for direct measurement of the reaction as fluorescent signals during the amplification process. amplification occurs, providing real-time data on the accumulation of PCR products. 3 Quantitative Analysis 4 Applications The fluorescence intensity is directly proportional to the qPCR has numerous applications in various fields, including amount of PCR product, enabling quantitative analysis of gene expression analysis, pathogen detection, and genetic target DNA or RNA. diagnostics. preencoded.png Real-Time PCR Chemistries: TaqMan Probes TaqMan Probe Mechanism Advantages of TaqMan Probes Disadvantages of TaqMan Probes TaqMan probes are oligonucleotide probes TaqMan probes offer high specificity and The primary disadvantage of TaqMan labeled with a fluorescent reporter dye sensitivity, allowing for accurate probes is their cost. They are more and a quencher dye. The quencher dye quantification of target sequences. They expensive to synthesize compared to absorbs the fluorescence emitted by the are also well-suited for multiplex assays, other qPCR chemistries. However, their reporter dye when they are close together. where different probes can be used to reliability and versatility make them a detect multiple targets in the same valuable tool for many applications. reaction. During PCR, the probe hybridizes to the target DNA sequence. The 5' nuclease activity of Taq polymerase cleaves the probe, separating the reporter and quencher dyes. As the reporter dye is released from the quencher, it emits fluorescence that is detected by the instrument. preencoded.png  TaqMan Probes Depend on Förster Resonance Energy Transfer (FRET) – inhibition of one dye caused by another without emission of a proton R – reporter Q- quencher. The reporter dye is found on the 5’ end of the probe and the quencher at the 3’ end  TaqMan Probes These probes are designed to hybridize to an internal region of a PCR product In the unhybridized state, the proximity (closeness) of the fluor (reporter) and the quench molecules prevents the detection of fluorescent signal from the probe Unhybridized Probe  TaqMan Probes Use DNA Polymerase to hydrolyse an oligonucleotide that is hybridized to the target amplicon Away from the quenching dye, the light emitted from the reporter dye in an excited state can now be observed Hybridized Probe TaqMan Probes  TaqMan Probes Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage Well-designed Taqman probes require very little optimization Can be used for multiplex assays by designing each probe with a spectrally unique fluor/quench pair Expensive to synthesize Real-Time PCR Chemistries: SYBR Green Mechanism SYBR Green is a dye that binds to double-stranded DNA. Upon excitation, SYBR Green emits fluorescence, which increases as the PCR product accumulates during amplification. Advantages SYBR Green is cost-effective and easy to use. It requires minimal optimization and can be used for detecting various targets with appropriate primers. Disadvantages SYBR Green can bind to any double-stranded DNA, including non-specific products, leading to overestimation of target concentration. It is also less sensitive than other chemistries like TaqMan probes. Applications SYBR Green is commonly used for relative quantification of gene expression and for screening PCR products for the presence of specific sequences. preencoded.png preencoded.png Real-Time PCR Chemistries: Other Options Chemistry Description Advantages Disadvantages Molecular Beacon Hairpin-shaped probe that High sensitivity, excellent for More complex than TaqMan fluoresces only when bound to multiplex assays. probes, requires careful target DNA. optimization. Scorpion Probe Combines a primer and a probe, Improved sensitivity and Requires careful design and reducing the need for separate specificity, cost-effective. optimization. probes. Amplifluor Direct Uses a single molecule that binds Simple and easy to use, cost- Lower sensitivity than other to the target DNA and emits effective. methods, can suffer from fluorescence upon binding. background noise. Plexor Similar to TaqMan probes, but High sensitivity and specificity, Expensive to synthesize, requires uses a different fluorophore. well-suited for multiplex assays. specialized instruments. LUX Uses a fluorescent dye that is High specificity, minimal Requires specialized instruments activated only upon extension of background noise. and reagents. the PCR product. preencoded.png Criteria for a Good qPCR Assay Specificity A good qPCR assay should produce only one specific product, ensuring that the fluorescence signal is solely due to the target sequence. Efficiency The reaction efficiency should be close to 100%, indicating that each cycle produces a doubling of the PCR product. Reproducibility The results should be reproducible, with consistent values obtained across multiple runs of the same experiment. Sensitivity A high sensitivity assay can detect very low levels of the target sequence, allowing for accurate quantification of even small amounts of DNA or RNA. preencoded.png Improving Reproducibility in qPCR Cleanliness A clean laboratory environment is essential for preventing contamination and ensuring accurate results. Work under a fume hood and use clean benchtop surfaces. Pipetting Technique Accurate pipetting is crucial for precise reagent dispensing. Use calibrated micropipettors and aerosol-resistant tips to minimize errors. Volume Optimization Use sufficient volumes of reagents to ensure accurate and consistent results. Consider preparing master mixes to minimize pipetting steps and reduce variation. Pipetting Consistency Pipette into each reaction vessel only once to minimize the risk of introducing variability and ensure consistent reagent concentrations. preencoded.png Conclusion: Real-Time PCR: A Powerful Tool Real-time PCR has revolutionized molecular biology, providing a sensitive and versatile tool for quantifying DNA and RNA. By understanding the principles, chemistries, and optimization techniques, researchers can harness the power of qPCR to answer important questions in various scientific disciplines. preencoded.png Advances in Lab-on-a- on-a-chip Technology Technology Dr Maimunah Mustakim preencoded.png  LEARNING OBJECTIVES AT THE END OF THE LESSON, STUDENTS ARE ABLE TO EXPLAIN/DESCRIBE/DISCUSS: Lab-on-a-chip (LOC) technology is revolutionizing diagnostics by miniaturizing laboratory processes onto microchips, enabling rapid, portable, and precise analyses. preencoded.png Introduction LOC systems integrate microfluidic channels, sensors, and actuators on a single chip, creating a miniature laboratory for biological and chemical analyses. 1 Miniaturization LOC devices require significantly smaller sample volumes, reducing costs and minimizing reagent consumption. 2 Integration They combine multiple laboratory functions into a single platform, enabling streamlined workflows and automated analysis. 3 Portability Compact size and ease of operation allow for point-of-care diagnostics, bringing testing closer to patients. 4 Precision Microfluidic control provides precise fluid handling and measurement, enhancing sensitivity and accuracy of analyses. preencoded.png Key Components of Lab-on-a-chip Technology LOC technology relies on three key components, each playing a crucial role in the miniaturization and automation of laboratory processes. Microfluidics Sensors and Detectors Automation Microfluidics is the manipulation of Integrated sensors, such as optical, LOC devices can be programmed to small volumes of fluids in electrical, or electrochemical sensors, perform automated tasks, including microchannels, enabling precise detect and measure analytes or sample preparation, reagent control over fluid flow, mixing, and changes in the microfluidic dispensing, and data analysis, reaction conditions. environment. minimizing human intervention and errors. preencoded.png Applications in Diagnostics LOC technology is transforming various fields of diagnostics, enabling rapid, accurate, and cost-effective analyses in a wide range of applications. Application Description Infectious Disease Detection Rapid detection of pathogens like viruses and bacteria using PCR or other molecular assays. Cancer Diagnostics Analysis of circulating tumor cells, detection of cancer biomarkers, and genetic screening for cancer predisposition. Genomics and Proteomics High-throughput analysis of gene expression, protein levels, and DNA sequencing for personalized medicine. preencoded.png Advantages of Lab-on-a-chip Technology LOC technology offers several advantages over traditional laboratory methods, making it a valuable tool for diagnostics and research. Miniaturization Reduced sample and reagent volumes, leading to cost savings and minimal waste generation. Portability Compact design allows for point-of-care testing, bringing diagnostics to remote or resource-limited settings. Speed and Automation Integrated sample preparation, analysis, and data interpretation result in faster turnaround times and reduced human error. Precision Microfluidic control ensures accurate fluid handling and measurement, enhancing the sensitivity and reliability of analyses. preencoded.png Recent Advances Constant research and development drive the advancement of LOC technology, leading to new capabilities and applications. Integration of Nanotechnology Nanomaterials like nanoparticles and nanowires are being incorporated into LOC devices to enhance sensitivity and improve detection limits. 3D Printing of Microfluidic Devices Advances in 3D printing allow for rapid and cost-effective fabrication of customizable microfluidic chips with complex geometries. Organ-on-a-chip Models LOC technology is being used to develop organ-on-a-chip models, simulating human organs and tissues in vitro for drug testing and disease modeling. preencoded.png Challenges and Future Directions Despite the significant progress, there are still challenges and opportunities for further development in LOC technology. Challenges Future Directions 1. Manufacturing complexity and cost. 1. Integration of artificial intelligence and machine learning. 2. Regulatory approval for clinical applications. 2. Development of advanced materials and fabrication 3. Standardization and interoperability of devices. techniques. 3. Expansion of applications in personalized medicine and diagnostics. preencoded.png Conclusion Lab-on-a-chip technology holds tremendous promise for transforming diagnostics and healthcare, enabling faster, cheaper, and more accessible tests. Rapid Diagnosis Point-of-Care Testing Reduced Costs Improved Accuracy Enables quicker diagnoses and Brings diagnostics to patients' Minimizes reagent consumption Provides more precise and reliable intervention, improving patient bedside, increasing convenience and laboratory expenses, making results, leading to more informed outcomes. and access. diagnostics more affordable. medical decisions. preencoded.png Impact on Healthcare LOC technology is poised to revolutionize healthcare, offering a range of benefits for patients, clinicians, and the healthcare system as a whole. Early Diagnosis 1 Early detection of diseases through rapid, accessible testing, leading to timely interventions and improved outcomes. 2 Personalized Medicine Tailored treatment plans based on individual genetic and molecular profiles, improving treatment effectiveness and Remote Monitoring 3 reducing side effects. Continuous monitoring of patients' health parameters in real-time, enabling proactive interventions and improved disease management. 4 Decentralized Healthcare Expansion of healthcare services to underserved communities, making diagnostics accessible to individuals regardless of their Research and Development 5 location. Accelerated drug discovery, personalized medicine research, and development of novel diagnostics for various diseases. preencoded.png Future Prospects The future of LOC technology holds immense potential for advancing healthcare and transforming the way we approach diagnostics, treatment, and disease prevention. Miniaturization and Widespread Adoption Wearable and Implantable Research and Innovation Integration Devices Increased adoption of LOC Continued research and Further miniaturization and technology across healthcare Development of wearable and innovation in LOC technology will integration of functionalities will settings, from hospitals to clinics implantable LOC devices for drive the development of new lead to even more compact and to home-based care. continuous health monitoring applications and advancements versatile LOC devices. and early disease detection. in diagnostics, drug discovery, and personalized medicine. preencoded.png PCR Point-of-Care: Revolutionizing Molecular Diagnostics Dr Maimunah Mustakim preencoded.png  LEARNING OBJECTIVES AT THE END OF THE LESSON, STUDENTS ARE ABLE TO EXPLAIN/DESCRIBE/DISCUSS: PCR point-of-care which refers to portable PCR devices that perform molecular diagnostics at the patient's location, offering rapid results and facilitating immediate decision-making in various healthcare settings. preencoded.png Key Features: Portability Convenience Portable PCR devices are easily transported to various locations, including clinics, field hospitals, and even homes, eliminating the need for sample transportation to a central laboratory. Accessibility Portability expands the reach of PCR diagnostics to underserved areas with limited access to centralized laboratories, facilitating timely diagnoses and treatments. Decentralized Testing The ability to test directly at the point of care eliminates the delay associated with sample transport and analysis, enabling rapid diagnosis and intervention. preencoded.png Key Features: Speed 1 Sample Collection The sample is collected using a swab or other methods appropriate for the specific diagnosis. 2 PCR Process The PCR process involves amplification of specific DNA or RNA sequences in the collected sample. 3 Result Interpretation The PCR results are interpreted by healthcare professionals to determine the presence or absence of the target molecule. preencoded.png Key Features: Ease of Use 1 User-Friendly Interface POC PCR devices are designed with intuitive interfaces that minimize the need for extensive training. 2 Simplified Procedures The testing process is simplified, often involving straightforward sample preparation and device operation. 3 Reduced Training Requirements Healthcare professionals with varying levels of experience can readily operate POC PCR devices, expanding access to molecular diagnostics. preencoded.png Key Features: Accuracy Traditional PCR POC PCR High sensitivity High sensitivity Accurate detection Accurate detection Reliable results Reliable results preencoded.png Applications: Infectious Diseases Rapid Detection Outbreak Management Personalized Treatment POC PCR can quickly identify In disease outbreaks, rapid and Knowing the specific pathogen infectious agents like bacteria, accurate diagnosis is crucial for causing an infection enables viruses, or parasites, allowing for containing the spread of infections healthcare professionals to tailor prompt isolation and treatment. and implementing appropriate treatments to the individual patient public health measures. and enhance treatment effectiveness. preencoded.png Applications: Genetic Testing Genetic Screening POC PCR can facilitate rapid genetic screening for inherited conditions, facilitating early diagnosis and intervention. Prenatal Testing POC PCR enables the detection of genetic abnormalities in fetuses during pregnancy, allowing for informed decision-making. Carrier Testing POC PCR facilitates carrier testing for recessive genetic disorders, enabling couples to make informed decisions regarding family planning. preencoded.png PCR Point-of-Care: The Future of Molecular Diagnostics Technological Advancements Constant innovation is driving the development of even more portable, user-friendly, and cost-effective POC PCR devices. Expanding Applications The scope of POC PCR is continually expanding to address a wider range of medical needs, including chronic disease monitoring and personalized medicine. Global Impact POC PCR holds immense potential for improving global health by facilitating accessible and rapid diagnostics in diverse settings. preencoded.png Gene Microarray Technology: Unraveling Gene Expression Patterns Dr Maimunah Mustakim preencoded.png  LEARNING OBJECTIVES AT THE END OF THE LESSON, STUDENTS ARE ABLE TO EXPLAIN/DESCRIBE/DISCUSS: Gene microarray technology that allows scientists to analyze gene expression patterns across thousands of genes simultaneously. This powerful tool has revolutionized the study of biological processes, including disease development and drug discovery. preencoded.png Microarray Basics Microarray Design Sample Preparation Data Analysis A microarray is a chip with a grid of DNA RNA from different samples is The fluorescent signals are scanned and probes, each representing a specific extracted, labeled with fluorescent analyzed to generate gene expression gene. The probes bind to dyes, and hybridized to the microarray. profiles. Statistical methods are complementary sequences in mRNA The intensity of fluorescence at each employed to identify genes that are samples, allowing for the quantification probe location represents the level of differentially expressed between of gene expression. gene expression. different samples. preencoded.png Applications of Microarray Technology Technology 1 Disease Diagnosis Microarray analysis can identify disease-specific gene expression signatures, aiding in the early diagnosis and prognosis of diseases like cancer. 2 Drug Discovery Microarrays help in identifying drug targets and assessing drug efficacy. This technology is crucial in the development of personalized medicine approaches. 3 Basic Research Microarrays provide valuable insights into complex biological processes, including development, aging, and response to environmental stimuli. 4 Agriculture Microarray technology is used to understand the genetic basis of crop yield and resistance to pests and diseases, leading to improved agricultural practices. preencoded.png Interpreting Microarray Data Green Lower Expression Red Higher Expression Yellow Similar Expression preencoded.png This microarray analysis profiled 18,000 genes expressed in normal and cancerous lymphocytes. The analysis was repeated to cover 1.8 million individual assays of gene expression. The malignant cells fall into two clusters. The orange cluster contains cells with expression profiles for GC B-like DLBCL cells. The blue cluster contains cells with expression profiles for activated B-like DLBCL cells. The cells within each cluster (shown across the top of the figure) are grouped by how closely their expression profiles resemble each other. The more closely the profiles resemble each other, the closer they are. The colors represent ratios of relative gene expression compared to normal control cells. Red represents expression greater than the mean level in controls, green represents expression lower than the mean level in controls, and the color intensity represents the magnitude of the difference from the mean.  Red fluorescence – highly expressed genes in cancer tissue samples Yellow - equal amount of expressed genes in normal and cancer tissue Green – expressed genes in normal tissues Clustering Analysis: Uncovering Hidden Relationships Data Preparation Microarray data undergoes normalization and transformation to ensure accurate comparisons. Clustering Algorithms Various algorithms, such as hierarchical clustering or K-means clustering, are used to group genes or samples based on similarity in their expression profiles. Cluster Interpretation The resulting clusters can reveal groups of genes that are co-regulated or samples that share similar biological states. preencoded.png Microarray Technology in Cancer Research Tumor Subtyping Microarrays help identify distinct subtypes of cancer based on their gene expression profiles, enabling personalized treatment approaches. Prognosis and Prediction Gene expression patterns can predict the likelihood of tumor recurrence or response to specific therapies, improving patient care. New Drug Targets Microarrays identify novel drug targets by revealing genes that are specifically expressed in cancer cells, leading to the development of more effective therapies. preencoded.png Advantages and Limitations of Microarray Technology Technology High Throughput Microarrays allow the simultaneous analysis of thousands of genes, providing a comprehensive view of gene expression. Sensitivity and Specificity Microarrays offer high sensitivity and specificity, allowing the detection of subtle changes in gene expression. Cost Microarray experiments can be expensive, limiting their accessibility to some researchers. Data Analysis Complexity Interpreting microarray data requires specialized bioinformatics skills, adding complexity to the workflow. preencoded.png Future Directions of Microarray Technology Future advancements in microarray technology include the development of smaller, more affordable platforms and the integration with other omics technologies, such as proteomics and metabolomics. This convergence will provide a more holistic view of cellular processes, leading to groundbreaking discoveries in medicine and beyond. preencoded.png