DNA SEQUENCING STUDY GUIDE
1. Manual DNA Sequencing Methods
a. Maxam–Gilbert (Chemical Cleavage)
- Cleaves DNA at specific bases using chemicals.
- Uses radioactive labels.
- Largely obsolete due to complexity and safety issues.
Mnemonic: "MAX is TOXIC"
- Maxam–Gilbert = Toxic chemicals and radioactivity.
b. Sanger Sequencing (Dideoxy Chain Termination)
- Incorporates ddNTPs (no 3’ OH) → stops DNA synthesis.
- Produces DNA fragments of varying lengths → electrophoresis.
- Each ddNTP is fluorescently labeled in automated versions.
Mnemonic: "SANGer STopS synthesis"
- Sanger uses Stopper ddNTPs to end the chain.
2. Automated Fluorescent Sanger Sequencing
- Uses cycle sequencing and fluorescent dyes (rhodamine,
fluorescein).
- Requires capillary electrophoresis.
- Software interprets the data via electropherograms.
Mnemonic: “Cycle + Colors = Sanger Automation”
3. Pyrosequencing
- Detects light emitted during nucleotide incorporation.
- Uses luciferase enzyme to generate luminescence.
- Based on PPi release → converted to ATP → light.
Mnemonic: “P-P-Pyro POPs Light”
- Pyrophosphate → Photon (light) → Pyrosequencing.
Best for: detecting known mutations and short sequences.
4. Bisulfite Sequencing (Methylation Analysis)
- Unmethylated cytosines → uracils (read as thymine).
- Methylated cytosines → stay cytosine.
- Used to analyze DNA methylation.
Mnemonic: "Bisulfite Bakes the C into U (unless Methylated)"
5. RNA Sequencing
- Captures mRNA via poly-A tails.
- Uses reverse transcription to cDNA before sequencing.
- Detects splice variants, editing, and expression levels.
6. Next-Generation Sequencing (NGS)
- Massively parallel sequencing: millions of reads in one run.
- Used for whole genomes, gene panels, and mutation profiling.
Steps:
1. Library Prep: Fragment DNA → add adapters.
2. Amplification: Emulsion PCR or bridge PCR.
3. Sequencing: Methods like:
- Reversible dye terminator
- Ion-conductance
- Sequencing by ligation
Mnemonic: “Next-Gen is L.A.S.T.”
- Library prep
- Amplify (PCR)
- Sequence
- Tools (bioinformatics)
7. NGS Platforms
a. Ion-Conductance
- Measures pH changes due to H⁺ release during nucleotide addition.
b. Reversible Dye Terminator
- Adds one fluorescently-labeled nucleotide at a time.
- Takes an image → remove dye → repeat.
c. Sequencing by Ligation
- Uses short, labeled oligonucleotides → 2 bases at a time.
Mnemonic: “Ion = H⁺, Dye = Click & Pic, Ligation = 2 by 2”
8. Key Terms
Term Definition
ddNTP - Dideoxynucleotide (chain terminator)
Polony - PCR colony on a flow cell (NGS)
Phred Score - Measures base-calling quality (20+ is good)
SNV/Indel/CNV - Types of sequence variants
Reference genome (hg19) - Used for alignment in NGS
High-Yield Mnemonic Summary
“MAX’s PYRO BURNED SANGER’s RNA, NEXT he built
SEQUENCERS by L.A.S.T.”
- MAX = Maxam–Gilbert
- PYRO = Pyrosequencing
- BURNED = Bisulfite (methylation)
- SANGER = Chain termination
- RNA = RNA sequencing
- NEXT = Next-Gen Sequencing
- L.A.S.T. = Library, Amplify, Sequence, Tools (Bioinformatics)
Test Tips
- Know which methods use fluorescence (Sanger, NGS) vs
luminescence (Pyrosequencing).
- Bisulfite sequencing distinguishes methylated vs unmethylated C.
- Reversible dye terminator is the most common NGS method.
- Sanger is ideal for short sequences, NGS for genome-wide
analysis.
