Detailed Notes on DNA Sequencing Techniques and Mutation Detection

 Mutation Detection Methods

 Amplification-Based Methods
 Principle:

Uses primers designed to target specific DNA regions.

 3’ end specificity: If the primer’s 3’ end matches the template DNA,

amplification occurs. If a mutation disrupts binding (e.g., a mismatch), no
amplification occurs.
 Steps:
 Design primers with 3’ ends complementary to the wild-type or suspected mutant
sequence.
 Perform PCR.
 Detect amplification via gel electrophoresis or real-time PCR.
 Applications:
 Detecting SNPs (single nucleotide polymorphisms), insertions, deletions.
 Example: Allele-specific PCR.
 Advantages:
 Simple, rapid, cost-effective.
 High specificity if primers are well-designed.
 Disadvantages:
 Limited to known mutations.
 Risk of false negatives if primer binding is inefficient.

 Restriction Fragment Length Polymorphism (RFLP)

 Principle:
 Mutations can create or abolish restriction enzyme recognition sites.
 Digested DNA fragments are analyzed via Southern blotting.
 Steps:
 Digest genomic DNA with a restriction enzyme.
 Separate dfragments via gel electrophoresis.
 Transfer to a membrane (Southern blot) and hybridize with a labeled probe.
 Detect fragment sizes using autoradiography.
 Applications:
 Diagnosing genetic disorders (e.g., sickle cell anemia).
 Advantages:
 Detects mutations without sequencing.
 High specificity.
 Disadvantages:
 Time-consuming (requires Southern blotting).
 Limited to mutations affecting restriction sites.

 High-Resolution Melting (HRM) Analysis

 Principle:
 Differences in DNA melting curves detect sequence variants.
 Homoduplexes (perfect matches) vs. heteroduplexes (mismatches) melt at
different temperatures.
 Steps:
 Amplify target DNA.
 Denature and reanneal to form homoduplexes (wild-type/wild-type or
mutant/mutant) or heteroduplexes (wild-type/mutant).
 Gradually increase temperature while monitoring fluorescence (using a dsDNA-
binding dye).
 Applications:
 Screening for SNPs, indels, and methylation changes.
 Advantages:
 Closed-tube method (no post-PCR processing).
 Detects unknown mutations.
 Disadvantages:
 Requires expensive instrumentation.
 Limited resolution in GC-rich regions.

 ---
 DNA Sequencing Techniques
 First-Generation Sequencing (Sanger Sequencing)
 Principle:
 Chain termination: Uses dideoxynucleotides (ddNTPs) to terminate DNA
synthesis randomly.
 Fragments are separated via capillary electrophoresis.
 Workflow:
 Template Preparation: Clone DNA fragments into vectors (e.g., plasmids).
 PCR Amplification: Generate DNA copies with fluorescently labeled ddNTPs.
 Electrophoresis: Separate fragments by size; detect fluorescence to determine
sequence.
 Advantages:
 Gold standard for accuracy (error rate: ~0.001%).
 Long read lengths (~800–1,000 bp).
 Disadvantages:
 Low throughput (1 reaction = 1 sequence).
 Expensive for large genomes.

 Shotgun Sequencing & the Human Genome Project

 Shotgun Approach:
 Steps:
 Fragment genomic DNA randomly.
 Clone fragments into vectors (e.g., BACs, YACs).
 Sequence fragments and assemble overlaps computationally.
 Challenges:
 Repetitive regions complicate assembly.
 Relies on high-quality libraries and computational power.
 Human Genome Project (1990–2003):
 Milestones:
 2001: Draft sequences published (public consortium vs. Celera).
 2003: “Finished” sequence (99% of euchromatin, ~2.85 billion bp).
  2022: Telomere-to-telomere (T2T) consortium completed remaining 8%
(heterochromatin/repeats).

 Second-Generation Sequencing (Next-Generation Sequencing, NGS)

 Core Principles:
 Massively parallel sequencing: Millions of reactions simultaneously.
 Sequencing-by-synthesis (SBS): Detect nucleotides as they are
incorporated.
 Clonal amplification: Generate clusters of identical DNA fragments (via
PCR or bridge amplification).

 Pyrosequencing (454 Sequencing)

 Workflow:
 Emulsion PCR: Amplify DNA fragments on beads in oil-water emulsions.
 Sequencing:
 Flow nucleotides sequentially (A, T, C, G).
 Incorporation releases pyrophosphate → converted to light via luciferase.
 Light intensity correlates with number of nucleotides added.
 Advantages:
 Longer reads (~700 bp) compared to Illumina.
 Disadvantages:
 High error rate in homopolymer regions.
 Obsolete due to high costs.

 Illumina (Solexa) Sequencing

 Workflow:
 Library Preparation:
 Fragment DNA → ligate adapters with known sequences.
 Clonal Amplification:
 Bind DNA to a flow cell → bridge amplification to generate clusters.
 Sequencing-by-Synthesis:
 Use fluorescently labeled, reversible terminator ddNTPs.
 Image fluorescence after each cycle → cleave terminator → repeat.
 Advantages:
 Ultra-high throughput (billions of reads per run).
 Low cost per base (~$0.01 per Mb).
 Disadvantages:
 Short read lengths (50–300 bp).
 Difficulty sequencing repetitive regions.

 Third-Generation Sequencing
 PacBio SMRT (Single-Molecule Real-Time Sequencing)
 Principle:
 Zero-mode waveguides (ZMWs): Observe DNA polymerase incorporating
fluorescently labeled nucleotides in real time.
 Advantages:
 Extremely long reads (up to 100,000 bp).
 Detects epigenetic modifications (e.g., methylation).
 Disadvantages:
 High error rate (~15%) → requires high coverage for accuracy.

 Oxford Nanopore
 Principle:
 DNA strands pass through nanopores; changes in electrical current detect
nucleotides.
 Advantages:
 Long reads (up to 2 Mb).
 Portable (e.g., MinION device).
 Disadvantages:
 High error rate (~5–15%).
 Sensitive to environmental noise.
 ---

Here’s a comparative table summarizing the features of various

sequencing techniques including Sanger, Illumina, PacBio, and
Nanopore:

Feature Sange Illumina PacBio Nanopore

r
Read 800– 50–300 10–100 kb 10 kb–2 Mb
Length 1,000 bp
bp
Throughp Low Very High Moderate Moderate
ut
Error 0.001 0.1% 10–15% 5–15%
Rate %
Cost per High $0.01 $0.10– $0.10–
Mb $0.50 $0.50
Applicatio Small Whole Structural Field
ns target genomes, variants, sequencing
s RNA-seq epigenetics , long reads

 ---

 Key Takeaways
 Mutation Detection:
 Amplification-based methods and HRM are rapid but limited to
known/suspected variants.
 RFLP is reliable but labor-intensive.
 Sequencing Evolution:
 1st-gen (Sanger): Accurate but low-throughput.
 2nd-gen (Illumina): High-throughput, short reads.
 3rd-gen (PacBio/Nanopore): Long reads, real-time analysis.
 Human Genome Project:
 Pioneered shotgun sequencing and computational assembly.
  Highlighted challenges of repetitive regions and the need for advanced
technologies.

 ---

 Study Questions
 Why is the 3’ end of a primer critical in amplification-based mutation detection?
 How does HRM distinguish between homoduplexes and heteroduplexes?
 Compare the roles of bacterial artificial chromosomes (BACs) and shotgun
sequencing in the Human Genome Project.
 Explain why Illumina sequencing struggles with repetitive regions.
 What advantages do third-generation sequencers offer over Sanger and Illumina?

Comprehensive Notes on PCR Techniques & Applications

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1. Hot Start PCR

- Problem:
- Non-specific primer binding during PCR setup leads to primer dimers, non-
target amplification, and reduced yield.
- Proofreading enzymes degrade primers with 3' overhangs.
- Solution:
- Inhibit DNA polymerase activity at room temperature using:
- Antibodies/Aptamers: Bind to polymerase.
- Chemical modifications: Block enzyme activity.
- Activation: Heat (95°C) during initial denaturation releases inhibitors,
restoring polymerase function.
- Advantages:
 - Reduces non-specific amplification.
- Improves yield of target amplicons.
- Applications: Genotyping, sequencing, multiplex PCR.

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2. GC-Rich PCR
- Challenge:
- GC-rich regions (3 hydrogen bonds) resist denaturation and form secondary
structures (hairpins).
- Solutions:
- Additives:
- Betaine or DMSO: Disrupt hydrogen bonds, lower melting temperature
(Tm).
- PCR Conditions:
- Higher denaturation temperatures (98°C).
- Longer denaturation times.
- Applications: Amplification of high-GC templates (e.g., promoter regions).

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3. Nested PCR
- Principle:
- Two rounds of amplification:
1. Primary PCR: Outer primers amplify target (may include non-specific
products).
2. Nested PCR: Inner primers bind to the primary product, ensuring
specificity.
- Advantages:
- Eliminates non-specific amplification.
- Increases sensitivity for low-abundance targets.
- Disadvantages:
- Requires opening tubes after first PCR (contamination risk).
---

4. Touchdown PCR
- Principle:
- Stepwise annealing temperature reduction:
- Start with high annealing temperature (e.g., 64°C) to favor specific primer
binding.
- Gradually decrease temperature (e.g., 0.5°C/cycle) to improve efficiency
once target amplicons dominate.
- Advantages:
- Reduces non-specific binding in early cycles.
- Enhances yield in later cycles.

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5. Agarose Gel Electrophoresis

- Purpose: Visualize PCR products by size.
- Protocol:
- Gel concentration:
- 1–2% agarose for 200–2,000 bp fragments.
- Stains:
- Ethidium bromide: Carcinogenic but sensitive.
- SYBR Safe/SYBR Green: Safer alternatives.
- Analysis: Band size compared to DNA ladder.

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6. Real-Time PCR (qPCR)

- Principle: Monitor amplification in real time using fluorescence.
- Detection Methods:
- SYBR Green:
- Binds dsDNA; non-specific (detects all products).
- Melting curve analysis post-PCR distinguishes specific vs. non-specific
products.
 - TaqMan Probes:
- Oligonucleotide with 5' fluorophore and 3' quencher.
- 5'→3' exonuclease activity of polymerase cleaves probe, releasing
fluorescence.
- Multiplexing: Multiple probes with distinct fluorophores.

Here’s a comparative table summarizing the features of One-Step and Two-Step

RT-qPCR:

Feature One-Step RT-qPCR Two-Step RT-qPCR

Workflow Reverse transcription cDNA synthesis first,

+ PCR in the same then PCR
tube

Speed Faster Slower

Contamination Low (closed tube) Higher (tube handling)

Risk

RNA Input Consumes RNA cDNA can be

stored/reused

Flexibility Limited to gene- Choice of RT primers

specific primers (oligo-dT, random
hexamers)

Data Analysis
- Threshold Cycle (Ct): Cycle where fluorescence crosses threshold (exponential
phase).
- Standard Curve: Serial dilutions of known DNA quantify target concentration.
- Relative Quantification (ΔΔCt):
1. ΔCt: Ct(target gene) – Ct(housekeeping gene).
2. ΔΔCt: ΔCt(sample) – ΔCt(control).
3. Fold change = 2^(-ΔΔCt).
- Housekeeping Genes: GAPDH, β-actin (stable expression across conditions).
---

7. Digital PCR (dPCR)

- Principle:
- Partition sample into thousands of reactions (microfluidic chambers/droplets).
- Endpoint PCR: Amplification in partitions with target DNA.
- Quantification: Count positive partitions (Poisson correction for multiple
copies).
- Advantages:
- Absolute quantification without standard curves.
- Detects rare alleles (e.g., cancer mutations).
- Applications: Liquid biopsy, viral load quantification.

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8. Rolling Circle Amplification (RCA)

- Principle:
- Circular DNA template + φ29 DNA polymerase (high processivity).
- Strand displacement generates long concatemers.
- Advantages:
- Isothermal: No thermocycler needed.
- High yield (μg DNA from pg input).
- Applications:
- Whole-genome amplification.
- Diagnostics in resource-limited settings.

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Key Takeaways
1. Hot Start PCR prevents non-specific amplification by inhibiting polymerase at
low temps.
2. Nested/Touchdown PCR enhance specificity for challenging templates.
3. qPCR enables quantification via Ct values; TaqMan offers higher specificity
than SYBR Green.
4. Digital PCR provides absolute quantification, critical for low-abundance
targets.
5. RCA is a versatile isothermal method for amplifying circular DNA.

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### **Comprehensive Notes: Physical Health, Disease Mechanisms, and
Molecular Diagnosis**

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#### **1. Physical Health & Homeostasis**

- **Definition**: Physical health refers to the body’s ability to function
optimally, maintain homeostasis, and meet physiological demands.
- **Homeostasis**: Dynamic equilibrium where internal conditions remain
stable despite external changes.
- **Failure**: Leads to illness (e.g., diabetes, cardiovascular diseases).
- **Key Concept**: *Wellness physiology* depends on maintaining
homeostasis.

---

#### **2. Categories of Physical Diseases**

1. **Deficiency Diseases**: Caused by lack of essential nutrients (e.g., scurvy
from vitamin C deficiency).
2. **Physiological Diseases**: Dysfunction in body systems (e.g., hypertension,
diabetes).
3. **Hereditary Diseases**: Genetic mutations (e.g., cystic fibrosis, sickle cell
anemia).
4. **Infectious Diseases**: Pathogen invasion (e.g., COVID-19, tuberculosis).

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#### **3. Disease Mechanisms: Intrinsic vs. Extrinsic Factors**

- **Extrinsic Factors** (External Causes):
- Malnutrition (e.g., obesity from overnutrition).
- Pathogens (viruses, bacteria).
- Physical trauma (injuries, blood loss).

- **Intrinsic Factors** (Genetic/Epigenetic):

- **Chromosomal Abnormalities**:
- *Numerical*: Extra/missing chromosomes (e.g., Trisomy 21 → Down
syndrome; XXY → Klinefelter syndrome).
- *Structural*:
- **Deletion** (loss of chromosome segment).
- **Duplication** (extra segment → gene overexpression).
- **Inversion/Insertion/Translocation** (altered gene regulation).
- **Gene Mutations**:
- **Point Mutations**:
- *Silent* (no amino acid change).
- *Missense* (amino acid substitution; e.g., sickle cell anemia).
- *Nonsense* (premature stop codon).
- **Frameshift Mutations**: Insertion/deletion altering reading frame (e.g.,
Huntington’s disease).
- **Abnormal Gene Expression**:
- Epigenetic changes (DNA methylation, histone modification).
- Example: Lynch syndrome (DNA repair gene silencing → cancer risk).

---

#### **4. Molecular Diagnosis Techniques**

- **Nucleic Acid Extraction**:
- **Specimens**: Blood, tissue biopsies, saliva, CSF.
- **Methods**:
- **Organic Isolation**: Phenol-chloroform separation.
- **Inorganic (Salting-Out)**: High-salt precipitation.
- **Solid-Phase (Column)**: Silica membrane binding.
- **Magnetic Beads**: Targeted nucleic acid capture.
 - **RNA-Specific Considerations**:
- Use RNase inhibitors (e.g., DEPC-treated water).
- Avoid contamination (gloves, separate workspace).

- **Quantification & Quality Assessment**:

- **Spectrophotometry**:
- A260/A280 ratio (DNA: ~1.8; RNA: ~2.0).
- A260/A230 ratio (detects salts/organic contaminants).
- **Fluorometry**:
- Dyes (e.g., Hoechst 33258 for dsDNA; SYBR Green for RNA/DNA).
- **Tools**: Nanodrop (UV absorbance), Qubit (fluorometric specificity).

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#### **5. Key Genetic & Epigenetic Concepts**

- **Epigenetics**:
- **DNA Methylation**: Silences genes (e.g., MLH1 in Lynch syndrome).
- **Imprinting Disorders**:
- *Beckwith-Wiedemann Syndrome*: Altered IGF2/CDKN1C expression →
overgrowth.
- **Telomerase & Cancer**:
- TERT promoter mutations → unchecked cell division (e.g., glioblastoma).

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#### **6. Clinical Applications**

- **Karyotyping**: Visualizes chromosomal abnormalities (e.g., Down
syndrome).
- **DNA Sequencing**:
- Identifies mutations (e.g., Sanger sequencing for point mutations).
- **PCR & RT-PCR**: Amplifies DNA/RNA for pathogen detection (e.g.,
COVID-19 testing).

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#### **7. Summary Diagrams**
1. **Chromosomal Abnormalities**:
- Numerical (Trisomy 21), Structural (Translocation in chronic myeloid
leukemia).
2. **Gene Mutation Types**:
- Silent, missense, nonsense, frameshift.
3. **DNA Extraction Workflow**:
- Cell lysis → organic/inorganic separation → ethanol precipitation.

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#### **Glossary**
- **Homeostasis**: Internal stability through feedback mechanisms.
- **Epigenetics**: Heritable changes without DNA sequence alteration.
- **Aneuploidy**: Abnormal chromosome number (e.g., Trisomy 18).

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These notes synthesize the molecular basis of diseases, diagnostic techniques,

and genetic mechanisms, providing a foundation for understanding
pathophysiology and modern diagnostics.