Midterm Review

Week 1: The basics of Molecular Epidemiology

Human biology overview

Most human cells have a nucleus, which is the cell's control center. Inside the nucleus, there are 46 chromosomes or 23 pairs, each made of DNA.

From chromosome to DNA: the process

  1. Chromatin: inside the nucleus, DNA is wrapped around proteins called histones, which form a structure dubbed chromatin

  2. Nucleosome: The chromatin is then organized into nucleosomes, which look like beads on a string

  3. DNA: The DNA molecule that results from the nucleosome, carries the instructions that are necessary for carrying out life

  4. RNA: DNA is transcribed into RNA, which is then needed in protein synthesis.


Central Dogma Theory: a theory that asserts that genetic information only flows in one direction; from DNA → RNA or from RNA → protein

What is DNA Replication?

DNA replication is the process by which a cell copies its own DNA to make sure that its DNA is passed down to its daughter cells during cell division. Each DNA molecule consists of one original strand and one newly synthesized strand.

Key Players (enzymes) in Replication

Helicase: unwinds the double-stranded DNA

Primase: synthesizes short RNA primers to start the replication process

DNA Polymerase: adds nucleotides to the growing strand

  • This is polymerase 3 in prokaryotes

  • There are multiple types in eukaryotes

Ligase: Joins Okazaki (newly synthesized DNA fragments) fragments on the lagging strand

Topoisomerase: relieves tension from the supercoiling ahead of the DNA fork

Where Replication Starts, Happens, Regulated, and the Role of Telomeres

Prokaryotes: replication has one point of origin

Eukaryotes: replication has multiple points of origin along each linear chromosome; it occurs in the nucleus and is tightly regulated in the S phase of the cell cycle

What is Transcription? (DNA → RNA)

the process of creating RNA from the DNA template made from replication

The nitty gritty…

  1. The initiation: RNA polymerase binds to a specific DNA sequence called a promoter and begins unwinding the DNA

  2. Elongation: RNA polymerase moves along the anti-sense (template strand) while adding complementary RNA nucleotides In A 5' to 3' direction

  3. Termination: The polymerase reaches a terminator sequence, stops, and releases the new RNA strand


Prokaryotic Vs. Eukaryotic Transcription

Prokaryotic (simple and faster):

  • They use 1 type of RNA polymerase to create all of their RNA

  • A protein called a sigma factor helps RNA polymerase find the right starting point or the promoter

  • genes are grouped into operons which nears that a single mRNA piece can carry instructions for multiple proteins

    • this is polycistronic mRNA

  • Due to prokaryotes not having a nucleus, transcription and translation happen at the same time in the cytoplasm. The cell essentially turns the nearly made RNA into a protein immediately.

Eukaryotic (complex and compartmentalized

  • they use 3 different RNA polymerases (1,2,3) each with a specific job and type of RNA to make.

  • instead of a sigma factor, they need special proteins called transcription factors to help RNA bind to DNA

  • transcription happens in the nucleus whereas translation occurs in the cytoplasm

  • before mRNA can leave the nucleus, it must be modified for protection and accuracy like a 5’ cap for stability, poly A trail, or splicing (Junk)

What is Translation (RNA → Protein)

Translation is where ribosomes translate the genetic code through mRNA to assemble amino acids into a protein.

  1. Initiation: Transfer RNA (tRNA) brings the specific amino acids to the ribosome. the ribosome assembles at the start codon (AUG) in eukaryotes and at the 5’prime cap in prokaryotes

  2. elongation: tRNAs enter the A site, a peptide bond forms in the P site and the empty tRNA exits through the E site as the ribosome moves across the mRNA

  3. termination: When a stop codon is reached (UAA, UAG, UGA), then release factors trigger the release of the new protein the ribosome then disassembles.

The Significance: Translation is the primary site for gene expression regulation and determining which proteins a cell produces and how many. Some viruses use reverse transcription to turn RNA into cDNA. They also hijack translation to build more virus particles by using the host’s ribosome and tRNAs to translate their viral DNA

Some exceptions to note….

  • reverse transcription: RNA can be converted into cDNA in some viruses

  • RNA editing: Changes to the RNA sequence after transcription can occur

  • non-coding RNA: some genes encode functional RNAs (tRNA,rRNA, and microRNA) rather than just proteins.

  • prions: these can replicate by converting normal proteins into prion form.

Week 2: Introduction to Virology

We need viruses, they infect all living things, and the human genome is 8% made up of endogenous retroviruses (ancient viral sequences that are permanent parts of an organism, often mutated and unable to cause disease, but do influence evolution.

ex: the retrovirus endogenous avian retrovirus (EAV-hp) became integrated in the chicken genome and activates the expression of a gene (SLCO1B3), which leads to a blue pigment that some chickens may lay.

Viruses are obligate intracellular parasites that are passive agents outside a host.

  • Ancient Integration: Roughly 8% of the human genome is comprised of Endogenous Retroviruses (ERVs).

  • Beneficial Viruses: Some viruses are mutualistic, such as Polydnaviruses that protect wasp eggs by suppressing host immunity, or CThTV, which grants heat tolerance to plants.

  • Viral Structure:

    • Capsid: A protein shell built with "genetic economy" using many copies of a few proteins. It also determines the shape or symmetry. Symmetry is typically icosahedral (round) or helical (rod-shaped). It also contains specific proteins that help it attach/enter host cells.

    • Envelope: A lipid bilayer stolen from the host cell, containing viral glycoproteins for host recognition.

  • Taxonomy: Classified by the ICTV into Realm (-viria), Kingdom (-virae), Phylum (-viricota), Class (-viricetes), Order (-virales), Family (-viridae), and Genus (-virus).

  • Baltimore Classification: Groups viruses into seven categories based on genome type (e.g., Group I is dsDNA; Group IV is +ssRNA) and how they produce mRNA.

Week 3: Viral Pathogenesis

A cell must be both susceptible (have the right receptor) and permissive (can replicate the virus) for infection to occur. A resistant cell has no receptor for the virus to enter or attach to

  1. Entry: Attachment to specific receptors (e.g., ACE2 for SARS-CoV-2; sialic acid for Influenza) through random collisions by chance. Entry often occurs via receptor-mediated endocytosis (clathrin or caveolin-mediated)

  2. Genome Replication:

    • DNA Viruses: Most must enter the nucleus to use host machinery (Exception: Poxviruses replicate in the cytoplasm).

    • RNA Viruses: Most replicate in the cytoplasm (Exception: Influenza requires the nucleus for mRNA processing). They must encode their own RNA-dependent RNA polymerase (RdRp) because cells lack this enzyme.

  3. Assembly: the virus makes new particles from its newly made parts from its genome

    • DNA viruses assemble in the nucleus

    • RNA viruses assemble in the cytoplasm

  4. Release of the virions:

    • Lysis: Non-enveloped viruses burst the cell.

    • Budding: Enveloped viruses gradually exit through the membrane, acquiring their envelope.

  5. Innate Immunity: The body detects viral PAMPs using receptors like TLRs or RIG-I, triggering Interferons (IFN). Viruses use evasion strategies (like SARS-CoV-2 NSPs) to block these signals

Week 4 & 6: Viral Transmission

Epidemiology measures how viruses spread and impact populations.

  • Core Metrics:

    • Incidence: New infections over time.

    • Case Fatality Rate (CFR): Deaths divided by confirmed cases.

    • R0​ (Basic Reproduction Number): Secondary cases from one infection in a fully susceptible population. Formula: R0​=τ×cˉ×d (transmissibility × contact rate × duration).

    • Re​ (Effective Reproduction Number): Accounts for immunity and interventions (Re​=R0​×S/N).

    • Dispersion Parameter (k): Quantifies superspreading. A low k (< 1) indicates highly heterogeneous spread where a few people cause most infections.

  • Transmission Chain:

    • Incubation Period: Time from infection to symptom onset.

    • Generation Time: Time between infection events.

    • Serial Interval: Time between symptom onset of the infector and the infectee.

  • Routes of Spread:

    • Horizontal: Respiratory (droplets vs. aerosols), Fecal-Oral (Norovirus), Vector-borne (Dengue via mosquitoes), or Sexual.

    • Vertical: Mother to child (Zika, HBV).

    • Internal: Can be hematogenous (via blood) or neuronal (moving anterogradely or retrogradely along axons, like Rabies or HSV)

Week 7: Molecular Detection I

I. Overview of Detection Methods

  • Traditional (Old) Methods: Historically, detecting pathogens required growing them in cell cultures, which is time-consuming, costly, and impossible for many "hard to grow" organisms.

  • Modern (New) Methods: Current techniques focus on direct testing through DNA/RNA-based molecular assays or immunological tests.

II. Polymerase Chain Reaction (PCR)

  • Fundamentals: Developed by Kary Mullis in 1983, PCR is a revolutionary technique used to amplify a few copies of DNA into millions.

  • Core Components: Successful PCR requires a DNA template, specific primers, Taq DNA polymerase (originally from Thermus aquaticus), nucleotides (dNTPs), a buffer, and Magnesium ions (Mg²⁺) as cofactors.

  • The PCR Cycle:

    1. Denaturation: Heating to ~94°C to separate the double-stranded DNA.

    2. Annealing: Cooling to 30–60°C so primers can bind to the template; this temperature is critical for specificity.

    3. Extension: Heating to ~72°C where the polymerase adds nucleotides to build new strands.

  • Key Parameters: Primer design is essential and should aim for 18–25 bases in length, 40–60% GC content, and avoid self-complementarity.

III. Quantitative Real-Time PCR (qPCR)

  • Quantification: qPCR allows for real-time monitoring of DNA amplification using fluorescence.

  • Cycle Threshold (Ct Value): The Ct value is the cycle number where the fluorescence signal crosses a set threshold; a lower Ct value indicates a higher initial concentration of the target gene.

  • Fluorescence Strategies: Assays use either SYBR Green I (a non-specific dye) or TaqMan Probes (highly specific) to generate signal.

  • RNA Detection: To detect RNA targets (like SARS-CoV-2), RT-qPCR is used, involving a reverse transcription step to create cDNA; this can be done in a one-step (all reagents in one tube) or two-step process.

IV. Advanced and Portable Detection

  • CRISPR-based Detection (SHERLOCK): This method uses Cas13a for single-molecule nucleic acid detection, providing high sensitivity for viruses like Zika, Dengue, and SARS-CoV-2.

  • Paper-based Assays: Designed for point-of-care testing in resource-limited settings, these portable, low-cost discs use isothermal amplification (like LAMP or RPA) to provide a visual readout.

  • Immunological Detection: These assays detect either antigens (pathogen proteins) or antibodies (host immune response) using formats like ELISA or lateral flow assays (similar to home COVID tests).

V. Metrics of Success (The Three S's)

  • Sensitivity: The limit of detection and the true positive rate.

  • Specificity: The true negative rate and the ability to avoid cross-reactivity.

  • Simplicity: Considering the equipment needs, technical expertise required, cost, and time to result

Week 8: Molecular Detection II

I. Illumina Sequencing (Second-Generation)

  • Dominance: Illumina is currently the dominant NGS platform due to its extreme throughput and cost-effectiveness.

  • Workflow:

    1. Library Prep: DNA is fragmented and ligated with specialized adapters.

    2. Cluster Generation: Clonal clusters are created on a flow cell through bridge amplification.

    3. Sequencing by Synthesis: Fluorescently labeled reversible terminators are added one by one; a camera records the emission to identify the base.

  • Data Format: Raw data is stored in FASTQ files, which include sequence identifiers, the raw sequence, and Phred quality scores (Q-scores). A Q30 score indicates a 1 in 1,000 error probability.

II. NGS Data Analysis Pipeline

  • Pre-processing: This involves quality control (FastQC) and trimming away adapters or low-quality bases.

  • Alignment: Raw "reads" are mapped to a reference genome using tools like BWA or Bowtie2.

  • Variant Calling: Identifying differences between the sample and the reference, such as SNPs (single nucleotide polymorphisms) or indels (insertions/deletions).

III. Third-Generation Sequencing (Long-Read)

  • PacBio (SMRT Sequencing): Utilizes Zero-Mode Waveguides (ZMW) and circular consensus sequencing (HiFi reads) to achieve long reads (10k–25k bp) with high accuracy.

  • Oxford Nanopore Technologies (ONT):

    • Mechanism: Threads single-stranded DNA through a biological nanopore; as nucleotides pass, they disrupt an ionic current, and the unique patterns identify the bases.

    • Unique Features: Capable of ultra-long reads (>100 kb), direct RNA sequencing (no cDNA step), and detecting native base modifications like methylation.

    • Portability: Devices like the MinION are pocket-sized and USB-powered, enabling real-time viral surveillance in the field.

IV. Key NGS Applications

  • Whole Genome Sequencing (WGS): Comprehensive analysis of the entire genome.

  • RNA-Seq: Profiles the transcriptome to detect differential gene expression or novel transcripts.

  • Metagenomics: Sequences all DNA in a sample (e.g., wastewater or gut) to study complex microbial communities without culturing.

  • Single-cell Sequencing: Profiling individual cells within heterogeneous populations