DNA Sequencing Reviewer

DNA Sequencing Reviewer

1. WHAT IS DNA SEQUENCING?

DNA sequencing is the laboratory technique for determining the exact order of nucleotides (A, T, C, G) in a DNA molecule. It is considered the most definitive molecular method for identifying genetic lesions and the gold standard for diagnosing genetic disorders, cancer mutations, infectious agents, and inherited diseases.

In the molecular biology workflow: PCR = amplified → Gel Electrophoresis = visualized → Sequencing = specified sequence

2. GENERATIONS OF DNA SEQUENCING FIRST GENERATION SEQUENCING

Chemical-based methods. Three types: Maxam-Gilbert, Sanger, and Capillary Sequencing (CapSeq).

METHOD 1: MAXAM-GILBERT (1977)

Developers: Allan Maxam & Walter Gilbert (published February 1977)

Principle: Chemical degradation — chemicals alter/modify the base structure, then piperidine cleaves the DNA at those sites, producing fragments of different lengths.

Key concept: Sequences the original sense strand (5' to 3'), radioactively labeled at the 5' end.

Chemicals used (4 tubes):

Steps:

1. Radioactively label the 5' end of the DNA

2. Divide DNA into 4 tubes

3. Add base-specific chemicals (alter structure only — no cleavage yet)

4. Add piperidine → cleaves the DNA at modified bases

5. Evaporate piperidine (dry → alcohol → dry, repeated 2–3x)

6. Resuspend in formamide

7. Run on polyacrylamide gel (best for <500 bp; high resolving power)

8. Dry gel and expose to light (autoradiogram)

9. Read sequence

How to read the gel:

• Read from bottom (shortest/farthest) to top (longest/nearest well) = 5' to 3'

• If band is in G lane only → Guanine

• If band in G+A lane but NOT G lane → Adenine

• If band in C+T and C lanes → Cytosine

• If band in C+T lane but NOT C lane → Thymine

Why it's no longer used: Chemicals are hazardous — DMS is toxic & combustible, formic acid is an irritant, hydrazine is flammable.

METHOD 2: SANGER SEQUENCING (Chain Termination Method, 1977)

Developer: Frederick Sanger (published December 1977)

Principle: Instead of degrading bases, Sanger uses dideoxynucleotides (ddNTPs) that lack a 3'-OH group. When a ddNTP is incorporated, chain elongation stops (no OH for polymerase to add the next nucleotide).

Key difference from Maxam-Gilbert: Sequences the newly synthesized strand, not the original. The base is NOT degraded — it is still included in the fragment.

Reagents needed:

1. DNA template strand

2. Radiolabeled DNA primer

3. DNA polymerase

4. dNTPs (normal deoxynucleotides)

5. ddNTPs (dideoxynucleotides — the chain terminators)

6. Manganese (promotes equal/simultaneous nucleotide addition)

Deoxynucleotide vs. Dideoxynucleotide:

• dNTP: 3' position = OH group (allows chain to continue)

• ddNTP: 3' position = H group (no OH = chain terminates)

4 tubes used:

Reading the gel: Same rule — read bottom to top (5' to 3'). Each lane is specific to one base, making it easier to read than Maxam-Gilbert. The shortest fragment = first base in the sequence.

Advantage over Maxam-Gilbert: Each lane is base-specific (not paired), so reading is more straightforward and no toxic degrading chemicals are used.

METHOD 3: CAPILLARY SEQUENCING (CapSeq)

Uses the same principle as Sanger but with two key differences:

• Uses a narrow capillary tube instead of a flat gel

• Has fluorescent dye incorporated into the ddNTPs → generates a light signal upon fragment formation → automatic detection, no manual reading needed

3. NEXT GENERATION SEQUENCING (NGS) NGS Types by Target Template:

2ND GEN: PYROSEQUENCING

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

Reaction mix: ssDNA template, primer, polymerase, nucleotide, APS + sulfurylase, luciferase + luciferin

How it works:

1. Polymerase extends the primer; each nucleotide added releases pyrophosphate (PPi)

2. PPi + APS → ATP (via sulfurylase)

3. ATP + luciferin → light signal (via luciferase)

4. Light pattern is detected and translated to sequence

Best for: Short-read tasks — detecting SNPs, mutations, DNA methylation
Not suitable for: Whole-genome or novel sequence discovery
Status: Largely discontinued due to toxic chemicals, but still used by some researchers

2ND GEN: ILLUMINA (Most widely used in PH)

Principle: Sequencing by Synthesis (SBS) — massively parallel, reads millions of fragments simultaneously

5 Basic Steps:

Step 1 – Fragmentation: DNA is cut into small pieces while still double-stranded

Step 2 – Library Preparation: Adapters are ligated to both ends of each fragment. Adapters = binding sites for primers + identification labels. Then denaturation separates strands into ssDNA.

Step 3 – Amplification (Bridge Amplification):

• DNA fragments added to a flow cell with immobilized (fixed) primers

• Adapters bind to complementary primers on the flow cell

• Polymerase extends → new strand made → original strand washed away

• Newly synthesized strand bends and connects to another nearby primer → forms a bridge

• Polymerase extends again → bridge is denatured → 2 fixed strands remain

• Process is repeated until sufficient copies are made (clusters)

Step 4 – Sequencing:

• Reverse (blue) strands are cleaved and washed away; only forward (red) strands remain

• Complementary strand is synthesized; fluorescent-labeled ddNTPs generate color signals:

Step 5 – Data Analysis: Signals are scanned and analyzed to determine the sequence

3RD GEN: PACBIO SEQUENCING

• Uses a hairpin adapter (SMRTbell template)

• Still fluorescence/signal-based like other SBS methods

• Correct nucleotide incorporation → fluorescence → sequence determined

4TH GEN: OXFORD NANOPORE SEQUENCING

Principle: Instead of light/fluorescence, uses electrical current changes

How it works:

• DNA molecule passes through tiny protein nanopores embedded in a membrane

• A constant electrical current flows through the pore

• Each nucleotide that passes through disrupts the current in a unique pattern

• The pattern of electrical changes identifies the nucleotide

• Sequence is determined from the unique current signatures

Key distinction: All other methods use light/fluorescence signals; nanopore uses electrical (ionic current) changes — this is why it's considered a separate generation entirely.

4. QUICK COMPARISON TABLE

5. HIGH-YIELD POINTS FOR THE EXAM

• Maxam-Gilbert sequences the original strand; Sanger sequences the new strand

• In Maxam-Gilbert, the targeted base is degraded and excluded; in Sanger, the base terminates the chain but is still included in the fragment

• Piperidine in Maxam-Gilbert = the cleavage agent (chemicals only modify, not cut)

• ddNTPs have no 3'-OH → that's why they terminate the chain

• Gel is read bottom to top (shortest fragment = first base = 5' end)

• Polyacrylamide gel is used (not agarose) because it handles fragments <500 bp with high resolution

• Illumina uses bridge amplification on a flow cell

• Nanopore is unique because it uses electrical current, not light

• Metagenomic sequencing = used when you don't know the target gene and want to see all organisms present