DNA Sequencing Reviewer (HY)

DNA Sequencing Reviewer (HY)

GOLDEN RULES TO MEMORIZE

• PCR = Amplified | Gel Electrophoresis = Visualized | Sequencing = Specified sequence

• DNA sequencing = gold standard for diagnosing genetic disorders, cancer mutations, infectious agents, inherited diseases

• Read gel always bottom to top = 5' to 3' direction (shortest fragment first = first base)

• Polyacrylamide gel is used (not agarose) → best for <500 bp, high resolving power

• Sequencing only needs one strand (complementary strand is inferred by base pairing rules)

FIRST GENERATION: THE BIG 3

MAXAM-GILBERT vs. SANGER — THE MOST TESTED COMPARISON

MAXAM-GILBERT: HIGH-YIELD FACTS

4 Tubes and their chemicals — MEMORIZE:

Step order to remember:
Radioactive label → 4 tubes → chemicals modify bases → piperidine cleaves → evaporate piperidine → resuspend in formamide → polyacrylamide gel → read autoradiogram

How to identify bases on the gel:

• Band in G lane only → Guanine

• Band in G+A lane, absent in G lane → Adenine

• Band in both C+T and C lanes → Cytosine

• Band in C+T lane, absent in C lane → Thymine

• The G lane (tube 1) acts as a confirmatory lane for the G+A lane (tube 2)

• The C lane (tube 4) acts as a confirmatory lane for the C+T lane (tube 3)

SANGER: HIGH-YIELD FACTS

Why "chain termination"?
ddNTPs have hydrogen (H) at both 2' and 3' positions — no hydroxyl group at 3' means polymerase cannot add the next nucleotide → chain stops

Normal dNTP: 3' = OH group → chain continues
ddNTP: 3' = H group → chain terminates

4 Tubes — simple and base-specific:

Key advantage over Maxam-Gilbert: Each lane is dedicated to one base only → easier to read, no confirmation lanes needed, no toxic degrading chemicals

Extra reagent: Manganese → promotes equal/simultaneous nucleotide addition (avoids consecutive addition bias)

CAPILLARY SEQUENCING (CapSeq): HIGH-YIELD FACTS

Same principle as Sanger — two differences only:

1. Uses a capillary tube instead of flat polyacrylamide gel

2. Has fluorescent dye on ddNTPs → automatic detection → no manual reading

NEXT GENERATION SEQUENCING (NGS): HIGH-YIELD FACTS NGS TYPES — KNOW WHAT EACH TARGETS:

PYROSEQUENCING: HIGH-YIELD FACTS

Principle: Sequencing by Synthesis (SBS) — light produced when correct nucleotide is incorporated

Reaction chain to memorize:
Nucleotide incorporated → PPi released → PPi + APS → ATP (sulfurylase) → ATP + luciferin → Light signal (luciferase) → sequence detected

Best for: SNPs, mutations, DNA methylation (short reads only)
NOT for: Whole-genome or novel sequence discovery
Status: Largely discontinued (toxic chemicals) but still used by some

ILLUMINA: HIGH-YIELD FACTS

Most widely used NGS — also an SBS method

5 Steps in order:

1. Fragmentation — DNA cut into small pieces while still double-stranded

2. Library Preparation — adapters ligated to both ends; adapters = primer binding sites + labels; then denatured into ssDNA

3. Bridge Amplification — DNA added to flow cell with immobilized primers; adapters bind → polymerase extends → original strand washed away → strand bends to form a bridge → extended again → repeated until clusters form

4. Sequencing — reverse (blue) strands removed; forward (red) strands sequenced; fluorescent ddNTPs generate color-coded signals:

5. Data Analysis — signals scanned and translated to sequence

Key term: Bridge amplification = unique to Illumina

PACBIO (3RD GEN): HIGH-YIELD FACTS

• Uses a hairpin adapter (called SMRTbell template)

• Still fluorescence-based like other SBS methods

• Correct nucleotide → fluorescence → sequence determined

• Platforms: PacBio Revio and PacBio Sequel

OXFORD NANOPORE (4TH GEN): HIGH-YIELD FACTS

The completely different one — uses ELECTRICAL CURRENT, not light

How it works:

• DNA passes through tiny protein nanopores in a membrane

• A constant electrical current flows through the pore at all times

• Each nucleotide disrupts the current in a unique pattern

• Pattern of electrical changes = identity of the nucleotide = sequence

Why it's 4th generation: All other methods = light/fluorescence; nanopore = ionic current changes → fundamentally different detection mechanism

MASTER COMPARISON TABLE

LAST-MINUTE MNEMONICS

Maxam-Gilbert chemicals in order (G, G+A, C+T, C):
"Dogs Fight Hungry Hyenas" → DMS, Formic acid, Hydrazine, Hydrazine+salt

Sanger tubes in order:
"All Cats Go There" → ddATP, ddCTP, ddGTP, ddTTP

Illumina color code:
"Big Orange Gorillas Run" → Blue=T, Orange=A, Green=G, Red=C

NGS generations:
"People In Philippines Need sequencing" → Pyrosequencing, Illumina, PacBio, Nanopore