MLS 1004SEF Intro to Mol Dx Lecture Notes PDF

Introduction to Medical Laboratory Science and Laboratory Instrument II (Course code: MLS 1004SEF) Introduction to Molecular Diagnostics 14:00 - 17:00 Thursday, 14 March 2024 Dr Kelvin Y.K. Chan [email protected] Outlines Definition of molecular diagnostics Specimen specification and processing; n...

Introduction to Medical Laboratory Science and Laboratory Instrument II (Course code: MLS 1004SEF) Introduction to Molecular Diagnostics 14:00 - 17:00 Thursday, 14 March 2024 Dr Kelvin Y.K. Chan [email protected] Outlines Definition of molecular diagnostics Specimen specification and processing; nucleic acids extraction for molecular diagnosis Common instruments and methods used for nucleic acids detection Laboratory design and general practice for molecular genetics workflow Other diagnostic tools and technologies 2 Timeline of the principal discoveries in the field of molecular biology, which influenced the development of molecular diagnostics 3 Comprehensive Role of Diagnostics Enable healthcare professionals (clinical doctors) to effectively oversee patient care across the entire spectrum of treatment 4 Definition of Molecular Diagnostics (Mol Dx) “Laboratory methods that are used to help identify a disease or the risk of developing a disease, such as cancer, by studying molecules, such as DNA, RNA, and proteins, in a tissue or fluid sample. Molecular diagnostics may also be used to help plan treatment for a disease, look for recurrence of disease, or find out how well treatment is working.” “There are many types of molecular diagnostic tests, such as biomarker tests, genetic tests, tumor sequencing tests, and liquid biopsies.” www.cancer.gov/publications/dictionaries/cancer-terms/def/molecular-diagnostics 5 The main categories of diagnostics: clinical chemistry, anatomical pathology, hematology, microbiology, and molecular diagnostics. Clinical Chemistry Hematology Anatomical pathology Medical microbiology Molecular Diagnostics Rare genetic disorders Immunogenetics Preimplantation genetic diagnosis Infectious diseases Oncology Prenatal Diagnosis Pharmcogenetics Transplantation genetics 6 Human haploid genome: ~3.3 G bp How about when it is diploid: ? bp Central dogma of molecular biology DNA (genome and genes), RNA (transcript), and proteins (amino acids) Functions of DNA Human genome (46 chromosomes) Express into RNA Translate into protein RNA Protein RNA  It serves as a store of information  ensuring that information is passed on to each new cell upon division (and the next generation)  It directs the synthesis of proteins  which are necessary to carry out the functions of a living organism Protein (20k-30k genes) 7 DNA Sequencing Sanger sequencing Frederick Sanger (MRC Centre, Cambridge, UK) published "DNA sequencing with chain-terminating inhibitors" in 1977 Since the early 1990s, DNA sequencing has almost exclusively been carried out with capillary-based, semi-automated implementations of the Sanger biochemistry. 8 Radioactively (P32) labelled ddNTP Fluorescently labelled ddNTP 9 DNA (Sanger) sequencing (fluorescent) Capillary electrophoresis-based semi-automated DNA Sequencer (e.g. ABI 3500xL) Template  Primer  G C T C A G C G A (either forward or reverse primer) Capillary Electrophoresis (CE) A G C G A C T C G  Advantages Long reads (~900bps) Suitable for small projects 384 reactions in parallel at most  Disadvantages Low throughput relatively expensive ($/bp) *Applied Biosystems (ThermoFisher) is the main technological provider 10 Sequencing of human genome 2001: Human Genome Project (2.7 billion, 11 years) 2001: Celera Corporation (100 million, 3 years) US dollar On April 1, 2022, Science published the first complete sequence of a human genome, known as T2T-CHM13. The challenging 8% of the human genome left unresolved by the initial Human Genome Project was sequenced and complete. Sequencing data and platforms: 30× PacBio circular consensus sequencing (HiFi), 120× ONT Nanopore ultralong-read sequencing, 100× Illumina PCR free sequencing, 70× Illumina Arima Genomics Hi-C, BioNano optical mapping, single-cell DNA template strand sequencing 11 Polymerase chain reaction (PCR) Amplicons detection or other subsequent nucleic acids analyses 12 Each PCR cycle includes three steps: (1)Denaturation of double-stranded DNA by heat; (2)Annealing of primers to their complementary target DNA sequences; (3)Extension of primers by a thermostable DNA polymerase. A typical PCR reaction is cycled 20–40 times Target size PCR reaction: Reaction buffer Primer pairs (forward & reverse) or (sense & antisense) dNTP Mg2+ Taq DNA polymerase DNA template (1) (2) (3) PMID: 29677144 13 1) In early PCR cycles, the signal due to amplification product remains at background level (baseline phase). The baseline is set to eliminate the background fluorescent signal. (2) During the exponential phase, the amount of PCR product doubles with each cycle (if amplification efficiency is 100%). The threshold line (dotted line) is set above the background within the exponential phase. The cycle of quantification (Cq) or threshold (Ct) is the cycle number at which the amplification plot intersects the threshold line that is set significantly above the baseline. A typical PCR amplification plot displays a sigmoidal-shape curve with 4 distinct phases (3) The linear phase indicates that reagents are becoming limited, which results in a reduction of the amplification efficiency. The amplification signal is no longer exponential. (4) The plateau phase indicates a saturation of the signal. Reagents are depleted and no additional PCR product is generated or detected PMID: 29677144 14 Nucleic acid amplification tests (NAATs)  Detection :  PCR/RT-PCR (analyzed by gel electrophoresis) PCR: polymerase chain reaction RT-PCR: reverse transcription PCR  starting material: DNA/RNA? RT-PCR  qPCR/qRT-PCR q: quantitative (real-time) RT PCR Extracted RNA  cDNA  targeted gene detection - Use of nucleic acid intercalating fluorescent dye (e.g. SYBR Green) Or - Targeting nucleic acids sequence hybridizing probe (e.g. TaqMan probe) Ct 15 16 Denaturation of dsDNA Primer annealing Primer extension Denaturation of dsDNA Primer and probe annealing Primer extension References: - IDT DNA. Real-Time PCR Handbook - Bonetta L. (2005). Prime time for real-time PCR. Nature Methods 2(4): 305–312. 17 Fluorescent dye-based qPCR Denaturation of dsDNA Primers annealing Primers extension Double stranded DNA (dsDNA) intercalating dye (or minor groove targeting dyes) show enhanced fluorescent signal upon binding With each amplification cycle, as the amount of the template dsDNA increases, the number of fluorophores bound, and therefore, the intensity of the fluorescent signal, increase. Examples of fluorescent intercalating dyes: SYBR® Green I and SYBR® Gold. Limitations: non-specific and bind all dsDNA and not just the target 18 Fluorophore-labeled sequence-specific probes Denaturation of dsDNA One of the popular types of probe: TaqMan probe The 5’-3’ exonuclease activity of Taq DNA polymerase to chew up the fluorophore-labeled sequence-specific oligonucleotide probe Primer and probe annealing Primer extension The oligonucleotide probe has a fluorophore (reporter dye) and a quencher at the 5’- and 3’- ends, respectively During primer extension, the exonuclease activity of the Taq DNA polymerase digests the probe which is hybridized to the targeting site downstream to the primer, hence separating the fluorophore from the quencher and resulting in increase of fluorescence. How do you determine the amount of nucleic acids from either methods? What kind of equipment? 19 Peltier (thermoelectric module) is a thermal control module that has both "warming" and "cooling" effects. By passing an electric current through the module, it is possible to change the surface temperature and keep it at the target temperature  for thermal cycling in PCR https://www.biobase.cc/Fluorescent-Quantitative-PCR-Detection-System-pd47650622.html 20 TaqMan probe The quencher molecule quenches (absorbs) the fluorescence emitted by the fluorophore (reporter dye) when excited by the light source via Förster resonance energy transfer (FRET) As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals  no fluorescent signal Free fluorodye, emit signal 21 Quantitative Real-time PCR assay using a standard curve. (a) Amplification curves for a 6-point 10-fold dilution series of a template with known concentrations (standard) over five orders of magnitude (e.g., genomic DNA, PCR amplicon, linearized plasmid). The Cq or Ct value of each serially diluted standard is determined; (b) A standard curve is generated by plotting the Cq or Ct values derived from the amplification curves of the dilution series against the logarithm of the standard quantity. The standard curve is used to interpolate the quantity of the target. The slope of the standard curve measures the amplification efficiency of the qPCR assay. (c) A slope of –3.32 (for a standard curve generated from a serial 10-fold dilution series) indicates 100% amplification efficiency, i.e., the amount of PCR product doubles during each cycle. 22 What is PCR and qPCR? https://www.youtube.com /watch?v=rpLSvEbOmqc Quantitative Real-time PCR (qPCR) https://www.youtube.com/watch?v=iu4s3Hbc_bw 23 Higher sensitivity & accuracy, and shorter turnaround time (TAT) Recent examples Rapid COVID-19 nucleic acid testing Significantly reduced the time to diagnosis in a couple of hours A new mycobacterium tuberculosis / rifampin (MTB/RIF) assay reduces the time to diagnosis of tuberculosis/rifampin resistant TB from 8 weeks to 2 hours and can be done “in the field”, in a mobile van, powered by solar energy 24 Workflow Specimen arrival  Nucleic acids preparation, amplification, and detection NA Extraction Specimen Detection PCR setup Nucleic acids (NA) workflow Amplification 25 Specimens FFPE Fine needle aspiration (FNA) Biological specimens or isolates Frozen tissue sectioning A) Anatomical pathology Amniocentesis Tumor tissue B) Clinical Chemistry C) Hematology Heel prick Peripheral blood D) Medical microbiology Bone marrow Bacterial isolate Nasal swab 26 Nucleic acids extraction Extractions of DNAs and RNAs In-house developed methods or commercial extraction kits Fast - can be finished in 20 minutes with consistent isolation conditions. Efficiency and reliable Use of non-toxic or less toxic reagents and no phenol chloroform 27 Manual vs Automation Low–throughput Laborious Work shifts High-throughput Less laborious 24 / 7 operation capability Higher flexibility Staff cost Less flexibility – standard workflow Capital cost 28 Laboratory design for nucleic acids amplification workflow 29 Molecular genomic/genetic lab setup and workflow design Mechanical barriers to prevent contamination Spatial separation of pre- and post-amplification work areas Area 1 – Reagent preparation Area 2 – Specimen/control preparation, PCR set-up Area 3 – Amplification/product detection, plasmid preparation Area 4 - Manipulation of amplified products Physically separated and, preferably, at a substantial distance from each other 30 Area 1 Area 2 Area 3 31 Unidirectional Workflow 32 Lab design – Example Using interlock hatch box (pass box) between rooms 33 Implementation of automated specimen processing station with closed-tube amplification and detection system 34 Features of each designated area 35 36 Other lab practices to prevent contamination and carry-over Minimal number of tube-to-tube transfer (witness) Use of positive displacement pipettes and disposable filtered pipette tips Avoid production of aerosols when pipetting Use of sterilized single-use plasticware Use of cleanroom sticky floor mats Minimizes the risk of amplicon carry-over on clothing, hair and skin Hairnet, dedicated safety glasses Disposable lab coat/gown and mask, color-coded preferred Gloves, need to change periodically and when working in different areas Shoe covers 37 Other lab practices to prevent contamination and carry-over Use of nuclease free or autoclaved water Aliquot oligonucleotides – multiple freeze thaws will cause degradation Always include a blank (no template) control to check for contamination Paperless workflow (electronic data system) Wipe test (swab test) Periodically (e.g. quarterly, monthly) Detect, localize, and remove contamination Identify the source of the contamination Clean and decontaminate the work area & equipment routinely Clean the PCR workstation at the start and end of each work day/run (UV light, fresh 10% sodium hypochlorite, DNA Away solution) Clean and decontaminate the exterior and interior parts of the pipette regularly 38 Type of Controls Internal Control Internal positive amplification controls to detect failure of DNA extraction or PCR amplification Reagent or equipment issues Integrity of DNA sample Presence of inhibitory substance External Control Positive control Negative control (normal, wild type) No template control (extraction blank) Blank 39 Internal controls (Abnormal allele) (Normal/wild type allele) *Positive amplification control: to ensure the amplification and reaction works; use different targeted gene or sequence NTC: no template control 40 Quality Controls Positive controls - expect to work under the given conditions - If your positive control does not work, those results indicate that something is wrong ? Inhibitors Sources of positive and negative controls: ? Reagents, thermal cycle condition Residual samples, EQA/PT* samples, ? Human mistakes QC materials: SeraCare, Corielle ? Machine or component failure *External quality assurance / proficiency test scheme Negative controls - Do not contain the target of your interest - Therefore, should not give you amplicons - If it does produce amplicons, might be a result of contamination or off-target amplification Blanks extraction control and No template controls To exclude nucleic acid contamination during extraction To exclude nucleic acid contamination during PCR 41 Allele drop-out (ADO) The failure of a molecular test to amplify or detect one or more alleles Potential causes: DNA template concentration Incomplete cell lysis DNA degradation Non-optimized assay conditions Unknown genetic variations present in target sites Reagent component failure Interfering substance Major concern for screening laboratories Confirmation of mutation inheritance in families may not an option Important: Primer and probe design (re-evaluation at least once a year for in-house developed nucleic acid amplification assay) to check whether variant(s) is/are reported within the primer sequence to avoid allele drop-out 42 False Amplification Potential causes: Non-optimized assay conditions Unknown nucleotide variations in target sites Gene duplications Oligonucleotide mis-priming (off-targeting) at similar sequence Pseudogenes or gene families Oligonucleotide concentrations too high Nucleic acids cross-contamination 43 Inherited (germline) vs acquired (somatic) variants Extracted from https://www.genomicseducation.hee.nhs.uk/cancer-genomics/ 44 Sources of genomic DNA for genetic testing Peripheral blood Heel prick Amniotic fluid Germline / inherited genetic variants Buccal/cheek swab Tumor tissue / biopsy Fine needle aspiration (FNA) Somatic / acquired genetic changes 45 Type of genetic variations - Short tandem repeats 46