Molecular Diagnostics Lecture Slides PDF

Introduction to Molecular Diagnostics COM5081 Fundamentals of Pathology Lecture 10 Jyotsna Chawla, Ph.D. Assistant Professor, Foundational Sciences, Dr. Kiran Patel College of Osteopathic Medicine, Nova Southeastern University. Objectives 1. Describe the molecular techniques in diagnosis: PCR, DNA...

Introduction to Molecular Diagnostics COM5081 Fundamentals of Pathology Lecture 10 Jyotsna Chawla, Ph.D. Assistant Professor, Foundational Sciences, Dr. Kiran Patel College of Osteopathic Medicine, Nova Southeastern University. Objectives 1. Describe the molecular techniques in diagnosis: PCR, DNA- Hybridization based assays, Next-generation sequencing (NGS) 2. Discuss the role of molecular diagnostics in diagnosing genetic disorders 3. Discuss the role of molecular diagnostics in cancer management/oncology 4. Discuss the role of molecular diagnostics in identifying infectious agents 2  Molecular Diagnostics Also known as Molecular Pathology, entails the examination of DNA or RNA, to identify specific disease indicators Applying molecular biology to medical testing A collection of techniques used to analyze biological markers in the genome and proteome  Investigates the human, viral, and microbial genomes, their genes, and the products they encode Source: https://cisncancer.org/research/new_treatments/molecular_diagnostics/importance.html 3 Milestones in Molecular Diagnostics 1953 Discovery of the DNA Double Helix James Watson and Francis Crick elucidated the structure of DNA 1983 Invention of Polymerase Chain Reaction (PCR) Kary Mullis pioneered a technique for amplifying targeted DNA sequences 1990-2003 Human Genome Project Complete sequencing of the whole human genome, including a comprehensive list of genes and their respective functions 2005-present Next-Generation Sequencing (NGS) DNA sequencing enables the examination of genomes, transcriptomes, and epigenomes 2008-present Pharmacogenomics & Personalized Medicine Genome-Wide Association Studies (GWAS) are conducted to identify genetic variations that are associated with diseases and drug response 4 Molecular Diagnostics: Sample Processing  Blood Samples: Whole blood, serum, or plasma for detection of infectious agents, and genetic markers  Saliva and Buccal Swabs (non-invasive): sample types are used for DNA-based genetic testing, including ancestry testing, pharmacogenomics  Tissue Biopsies: Tissue samples obtained from surgical resections or biopsies are important for molecular profiling in cancer diagnostics  Cell-Free DNA/RNA: Circulating cell-free DNA Conventional laboratory protocol of spin-column-based nucleic acid and RNA obtained from plasma or other extraction for molecular diagnostics for COVID-19. https://doi.org/10.1002/adma.202206525 bodily fluids are increasingly used for liquid biopsy, prenatal diagnostics  Other Samples: soil, water, food, may be  Key steps in sample processing: Sample collection, analyzed for the presence of microbial storage Quality control (purity of sample) pathogens and toxin contamination Optimized extraction method 5 Polymerase Chain Reaction (PCR) PCR is a molecular technique that amplifies a targeted DNA sequence, resulting in numerous copies of a given sequence Principle: It is based on the principle of DNA replication and uses a DNA template, primers, and a heat-stable DNA polymerase enzyme Method: 1. Denaturation- DNA is heated to separate the two strands 2. Annealing- primers specific to the target DNA sequence bind to the DNA 3. Extension- DNA polymerase synthesizes new DNA strands using the primers as a starting point 6 Polymerase Chain Reaction (PCR): Variations Reverse Quantitative PCR Conventional PCR Transcriptase PCR (qPCR) (RT-PCR) Amplify specific Detect RNA Quantify DNA sequences sequences DNA or RNA E.g., Gene Expression E.g., Genetic testing, Analysis (cancer, E.g., Viral Load Diagnosis of infectious autoimmune Monitoring (HIV, diseases (tuberculosis, disorders, and hepatitis B and C, and hepatitis, and sexually neurological COVID-19) transmitted infections) conditions) 7 Polymerase Chain Reaction (PCR): Advantages:  High sensitivity compared to culture and staining  Quickly performed in 4-8 hours  Rapid and cost-effective amplification of DNA  Ability to detect less common organisms such as viruses (able to amplify a single copy of a nucleic acid target, often undetectable by standard hybridization methods, and multiply to 107) Limitations:  Susceptible to contamination, which can lead to false-positive results  Limited to the amplification of relatively short DNA sequences  The potential for primer-dimer formation and non-specific amplification, primer design requires expertise 8 DNA Hybridization Assays DNA hybridization assays detect, locate, and quantify DNA or RNA sequences Principles of DNA hybridization Principle: Non-amplification-based detection that involves the annealing of single-stranded DNA or RNA probes to their complementary sequences in a sample Method: A labeled probe molecule with a sequence complementary to the target DNA or RNA is allowed to bind to the target sequence. The binding is detected by autoradiography, fluorescence, or chemiluminescence. Source: DOI:10.1109/TMBMC.2016.2537305 Advantages: Longer probes increase target specificity, which is useful when capturing a range of genetic variants (e.g., viral strains with high mutation rates) Limitations: Conditions for hybridization need to be optimized; labeled probes increase the cost of the assay 9 DNA Hybridization Assays Southern Blotting Northern Blotting  Detects sample DNA sequences  Detects sample RNA sequences  DNA fragments are transferred from the Agarose  DNA fragments are transferred from the Agarose gel to a solid substrate like nitrocellulose or nylon gel to nylon membrane and hybridized with tagged membrane and hybridized with tagged DNA probes DNA / RNA probes  Clinical use: Detection of genetic mutations- e.g.,  Clinical use: Level of expression of disease CFTR gene in cystic fibrosis; Triplet repeat disorders markers- e.g., HER2 in breast cancer e.g., Huntington's Disease 10 https://www.excedr.com/resources/western-vs-southern-vs-northern-blot DNA Hybridization Assays A typical FISH procedure Fluorescence in situ hybridization (FISH)  Detects and localizes the presence or absence of specific DNA sequences on chromosomes  Principle: Fluorescently tagged DNA probes that are complementary to the target DNA sequence bind to the DNA in the sample and produce a fluorescent signal, measured by a fluorescence microscope. The probe's fluorescence pattern reveals the presence, absence, or abnormality of the target DNA sequences  Advantages: High specificity, provides spatial information on the location of specific DNA sequences within cells and tissues  Limitations: Labor-intensive, time-consuming, and interpretation requires expertise  Clinical Use: Prenatal- detect chromosomal abnormalities such as aneuploidy (e.g., trisomy 21, 18, 13); Cancer - identify specific chromosomal rearrangements, gene amplifications, and deletions in Image source: https://www.researchgate.net/publication/235752267_Detection_of_Sa biopsy tissue lmonella_spp_Presence_in_Food 11 DNA Hybridization Assays Fluorescence in situ hybridization (FISH) Types of chromosome alterations detected by FISH Source: DOI:10.1186/s42047-021-00096-1 DNA Hybridization Assays Microarray  A powerful tool for analyzing gene expression, genetic variation, and genomic interactions.  Principle: can probe for thousands of different mRNAs simultaneously. The sample is labeled with a fluorescent or radioactive tag  Method: a chip (glass/silicone) has thousands of DNA sequences robotically attached in a grid array RNA is extracted cDNA fragments of the samples are labeled added to the chip a computer analyzes the degree of binding to various regions of the chip  The difference in the intensity of the colors for each spot determines the expression level of genes  Advantages: Allows simultaneous analysis of thousands of nucleic acid sequences in a single experiment  Limitations: Expensive, short-shelf life of DNA chips, analysis is complex  Clinical Use: Analyze cancer cell gene expression, Single Nucleotide Polymorphism (SNP) Genotyping, Copy Number Variation (CNV) Analysis 13 Next-generation Sequencing (NGS)  NGS is a high-throughput, parallel sequencing technology  Principle: Clonally amplified DNA clusters are generated on a solid surface, followed by sequencing-by-synthesis, which results in sequence data that is analyzed  Method: 1. Library preparation: DNA or RNA from the sample is fragmented, and adaptors are ligated to the ends of the fragments to enable amplification 2. Cluster generation: The DNA fragments are clonally amplified to create clusters on a solid surface, such as a flow cell or a sequencing chip. 3. Sequencing: The DNA clusters are sequenced using sequencing-by- synthesis 4. Data analysis: Sequencing reads are aligned, and data are interpreted 14 Next-generation Sequencing (NGS) The evolution of DNA sequencing tools 15 Molecular Diagnostics for Genetic Disorders Carrier Screening E.g., Cystic Fibrosis  Cystic fibrosis is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene  CFTR follows an autosomal recessive pattern of inheritance  CFTR Mutation Analysis is a whole CFTR gene sequencing test that can detect 5 genetic variants TruSight Cystic Fibrosis NGS Test  CFTR test is advised to determine whether individuals are carriers of a CF genetic mutation and to evaluate the risk of passing the mutation on to the offspring 16 Molecular Diagnostics for Genetic Disorders Prenatal Screening E.g., Chromosomal Abnormalities- Down's syndrome (Trisomy 21), Edward's syndrome (Trisomy 18) and Patau's syndrome (Trisomy 13)  Cellfree DNA Test: Non-invasive prenatal testing (NIPT) uses maternal serum to detect fetal genetic abnormalities; performed as early as 10 weeks  Amniocentesis (AC): Sampling of the amniotic fluid, performed between 10 -14 weeks; Miscarriage risk  Chorionic villus sampling (CVS): Sampling of the placenta, performed between 16 -18 weeks; Miscarriage risk Fluorescence in situ hybridization (FISH) can be used to detect common aneuploidies 17 Molecular Diagnostics for Genetic Disorders Metabolic disorders were previously Heel stick method for sample identified by the Guthrie Test- Newborn Screening collection bacterial inhibition assay Testing newborns for a panel of genetic, metabolic, and congenital disorders to identify conditions early E.g., Phenylketonuria (PKU)  Mutations in the phenylalanine hydroxylase gene cause PKU.  Metabolic disorder- Inability to metabolize the amino acid phenylalanine high levels of Phenylalanine in  Expanded Newborn Screening Using Tandem Mass blood severe cognitive impairment Spectrometry  Treatment: Phenylalanine-restricted diet, formula  Tandem Mass Spectrometry (MS/MS): Molecular Diagnostic technology that enables the identification and quantification of several metabolites  Principle: Sample molecules are ionized and then separated based on their mass-to-charge ratio identification of individual components with very high specificity  Increased capacity for screening; Florida's panel contains ~35 metabolites 18 Messina, M. et. Al. Int. J. Neonatal Screen. 2018, 4, 12. https://doi.org/10.3390/ijns4020012 Molecular Diagnostics in Oncology Early Detection & Diagnosis  Liquid biopsy: A blood tests that detect materials shed from a tumor such as circulating tumor DNA  A new diagnostic tool to better diagnose and assess cancer progression  Early detection of certain cancers-lung, breast, and colorectal cancer  Molecular tools like RT-PCR, NGS are used to https://www.nature.com/articles/s41571-020-00457-x analyze the sample and identify disease biomarkers 19 Molecular Diagnostics in Oncology Management E.g., Oncotype DX in breast cancer  Predicts disease recurrence and informs adjuvant therapy decisions E.g., MolDX: Minimal Residual Disease  Patients' circulating tumor DNA levels can be monitored to measure therapy response and discover residual illness  Used in hematopoietic malignancies 20 Molecular Diagnostics in Oncology Treatment  Targeted Molecular Therapy: Personalized medical therapy designed to treat cancer by interrupting unique molecular abnormalities that drive cancer growth  E.g., EGFR Testing for Non-Small Cell Lung Cancer: PCR, NGS detects mutations in the epidermal growth factor receptor (EGFR) gene  mutations are associated with a better response to EGFR  Overexpression of HER2 in breast cancer cells tyrosine kinase inhibitors (TKIs)  E.g., HER2 Testing for Breast Cancer: FISH is used to identify overexpression or gene amplification of the HER2 protein in breast cancer cells HER2 positive patients respond better to targeted therapies such as trastuzumab 21 Molecular Diagnostics in Infectious Diseases Pathogen Detection:  E.g., Polymerase Chain Reaction (PCR) and real-time (RT-PCR) are widely used to detect the presence of bacterial, viral, and fungal pathogens in clinical samples to identify the causative infectious  Viral Load Monitoring agents such as influenza, COVID-19, and tuberculosis Viral Load Monitoring:  E.g., Quantitative PCR (qPCR) is used to detect the presence of viruses in a patient's blood or bodily fluids, allowing for the monitoring of viral load in diseases such as HIV, hepatitis B, and hepatitis C.  This information is crucial for determining treatment efficacy and illness progression Source- Macchi B, et al. Appraisal of a Simple and Effective RT-qPCR Assay for Evaluating the Reverse Transcriptase Activity in Blood Samples from HIV-1 Patients. Pathogens. 2020; 9(12):1047. https://doi.org/10.3390/pathogens9121047 22  Molecular Diagnostics in Infectious Diseases Antimicrobial Resistance Testing: Molecular approaches can detect specific genetic markers associated with antimicrobial resistance, assisting with the selection of appropriate antimicrobial therapy  E.g., PCR assays can identify genes conferring resistance to antibiotics, such as the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) pathogens  Tracking antimalarial resistance  E.g., Molecular epidemiology- Targeted Amplicon Deep Sequencing (type of NGS) was utilized to detect Plasmodium falciparum kelch13 mutations associated with artemisinin resistance parasites. The distribution of resistance markers in the population can be monitored using these molecular tools. 23 Molecular diagnostics in Infectious Diseases  Sequencing COVID variants at Wellcome Sanger Institute, UK Genotyping and Strain Identification E.g., Identification of Coronavirus Variants Whole-genome sequencing was used to track viral outbreaks and strains during the COVID-19 pandemic E.g., Influenza Virus Vaccine Every year, the sequence of the gene segments of circulating influenza viruses is retrieved and put in databases, allowing researchers to predict antigens for vaccine production 24 References & Resources  Clinical Molecular Diagnostics. (2021). Singapore: Springer Nature Singapore  Valones, M. A., Guimarães, R. L., Brandão, L. A., de Souza, P. R., de Albuquerque Tavares Carvalho, A., & Crovela, S. (2009). Principles and applications of polymerase chain reaction in medical diagnostic fields: a review. Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology], 40(1), 1–11. https://doi.org/10.1590/S1517-83822009000100001  Prenatal Screening https://www.acog.org/womens-health/faqs/prenatal-genetic-screening- tests#:~:text=The%20cell%2Dfree%20DNA%20in,week%20to%20get%20the%20results.  Newborn Screening https://floridanewbornscreening.com/  Sokolenko, A. P., & Imyanitov, E. N. (2018). Molecular Diagnostics in Clinical Oncology. Frontiers in molecular biosciences, 5, 76. https://doi.org/10.3389/fmolb.2018.00076  HER2 Gene Amplification Testing by Fluorescent In Situ Hybridization (FISH) https://ascopubs.org/doi/10.1200/JCO.2016.66.6693  Liu, Q., Jin, X., Cheng, J., Zhou, H., Zhang, Y., & Dai, Y. (2023). Advances in the application of molecular diagnostic techniques for the detection of infectious disease pathogens (Review). Molecular medicine reports, 27(5), 104. https://doi.org/10.3892/mmr.2023.12991 25