DNA Sequencing

DNA sequencing is the process of determining the exact order of nucleotides (A, T, G, and
C) in a DNA molecule. It helps scientists:
 Identify genes and mutations,
 Understand genetic diseases,
 Trace evolutionary relationships,
 Improve crop and animal breeding,
 Develop personalized medicine.
There are different methods of sequencing, but Sanger sequencing (also called chain
termination method) was the first widely used technique and is still used for accurate,
small-scale sequencing (e.g., individual genes or PCR products).
What is Sanger Sequencing?
Sanger sequencing is a method developed by Frederick Sanger in 1977. It uses DNA
polymerase to synthesize new DNA strands, but it also uses special modified nucleotides
called dideoxynucleotides (ddNTPs) that terminate DNA strand elongation. This creates
DNA fragments of different lengths that can be used to determine the DNA sequence.
Step-by-Step Procedure of Sanger Sequencing
🧪 Step 1: Preparation of DNA Template
 A single-stranded DNA (ssDNA) template.
 Usually, this is obtained by amplifying the desired DNA using PCR (polymerase
chain reaction).
🧬 Step 2: Reaction Mixture Setup
In each sequencing reaction tube (or well), is added:
1. Template DNA – the DNA to be sequenced.
2. DNA Primer – a short sequence that binds (anneals) to the template to start synthesis.
3. DNA Polymerase – an enzyme that adds nucleotides to form a new DNA strand.
4. Normal deoxynucleotides (dNTPs) – A, T, G, and C for normal DNA synthesis.
5. Labeled dideoxynucleotides (ddNTPs) – Chain-terminating nucleotides that are:
o Missing the 3’-OH group (can’t extend DNA further),
o Fluorescently labeled with different colors for A, T, G, and C.
Only a small amount of ddNTPs is added so that DNA chains are terminated randomly at
different positions.
Step 3: DNA Synthesis and Chain Termination
 DNA polymerase starts synthesizing the complementary strand.
 When a ddNTP is randomly incorporated instead of a dNTP, the chain stops
growing.
 This process produces DNA fragments of varying lengths, each ending at a ddNTP.
For example, if a ddATP is added, synthesis stops at an A base.
Step 4: Fragment Separation by Capillary Gel Electrophoresis
 The DNA fragments are run through a capillary gel (thin glass tube filled with gel-
like material).
 Smaller fragments move faster than larger ones.
Step 5: Detection of Fragments
  As fragments exit the capillary tube, a laser detects the fluorescent labels on
ddNTPs.
 Each ddNTP emits a unique color, allowing the machine to read the final base in
each fragment.
For example:
 ddATP = green
 ddTTP = red
 ddGTP = yellow
 ddCTP = blue
Step 6: Sequence Analysis
 The computer collects the color signals and converts them into a chromatogram (a
colored graph).
 The chromatogram shows peaks for each base, and the DNA sequence is read from
smallest to largest fragment.
Summary of Sanger Sequencing Steps
Step Description
1. Prepare single-stranded DNA template
2. Add primer, DNA polymerase, dNTPs, and fluorescent ddNTPs
3. Perform DNA synthesis → Random chain termination occurs
4. Separate DNA fragments using capillary gel electrophoresis
5. Detect colored fragments using a laser
6. Read the sequence from fluorescence pattern

Key Features of Sanger Sequencing

 Highly accurate for short DNA sequences (up to ~1000 bp).
 Used for:
o Validating mutations,
o Sequencing PCR products or plasmids,
o Diagnosing genetic diseases.
Maxam–Gilbert sequencing
Maxam–Gilbert sequencing is a chemical cleavage method used to determine the nucleotide
sequence of DNA. It involves labeling the DNA at one end and then selectively breaking the
DNA strand at specific bases using chemical reactions. The resulting fragments are separated
by gel electrophoresis to deduce the sequence.
Steps of Maxam–Gilbert Sequencing
1. DNA Preparation
 Obtain purified double-stranded DNA.
 Denature it to make it single-stranded.
 You need only one strand of DNA for sequencing.
2. End-Labeling the DNA
 The 5′ end of the DNA is labeled using radioactive phosphorus (³²P).
o This is done using the enzyme polynucleotide kinase and γ-³²P-ATP.
 This labeling allows only the cleaved fragments containing the 5′ end to be
visualized on the gel.
3. Chemical Cleavage Reactions
The labeled DNA is divided into four separate reaction tubes, each treated with a chemical
that breaks the DNA at specific bases:
Tube Cleavage Target Chemicals Used
A+G Purines (A & G) Formic acid
G Guanine only Dimethyl sulfate (DMS)
C+T Pyrimidines (C & T) Hydrazine
C Cytosine only Hydrazine + high salt (NaCl)
 The chemicals modify specific bases.
 A cleaving reagent (usually piperidine) is added to break the DNA backbone at the
modified sites.
4. DNA Fragment Generation
 The result is a set of DNA fragments of varying lengths.
 Each fragment ends at a specific base that was cleaved chemically.
5. Gel Electrophoresis
 All four reactions are run side by side on a high-resolution polyacrylamide gel.
 The gel separates DNA fragments by size — shorter fragments migrate faster.
6. Autoradiography
 The gel is exposed to X-ray film to visualize the radioactively labeled fragments.
 A pattern of bands appears, each corresponding to a DNA fragment ending at a
specific base.
7. Reading the Sequence
 Start from the bottom of the gel (shortest fragments) and read upward.
 Compare the bands in the four lanes (G, A+G, C, C+T) to determine the DNA
sequence.
o For example, a band in G lane = Guanine.
o A band in A+G but not in G = Adenine.
Summary
 Accurate but labor-intensive
 Requires hazardous chemicals
 Largely replaced by Sanger sequencing and modern methods
 Useful in the early days of genome sequencing

NEXT-GENERATION SEQUENCING
After Sanger and Maxam–Gilbert sequencing methods, DNA sequencing technology has
advanced rapidly, improving speed, accuracy, cost-efficiency, and scalability. Here's an
organized overview of the major advances in sequencing technologies post-Sanger:
Next-Generation Sequencing (NGS) (2005 onwards)
NGS technologies massively parallelize the sequencing process — millions of fragments
are sequenced at once, unlike Sanger's single-fragment approach.
Key Features:
 High throughput
 Lower cost per base
 Short reads (50–300 bp)
 Suitable for whole-genome, exome, RNA, and metagenomic sequencing
Major Platforms:

Platform Key Technology Notes

Illumina Reversible terminator chemistry Most widely used
Ion pH-based semiconductor detection No optical systems used
Torrent
Roche 454 Pyrosequencing Discontinued; was early NGS
SOLiD Sequencing by ligation High accuracy but complex
2. Third-Generation Sequencing (TGS)
Unlike NGS, these allow real-time sequencing of single molecules without amplification.
Features:
 Long reads (up to hundreds of kb)
 Detects base modifications (e.g., methylation)
 More error-prone than NGS (but improving)

Technologies:
Platform Method Advantage
PacBio (SMRT) Real-time fluorescence Long reads, epigenetics
Oxford Nanopore Nanopore electrical Portable, ultra-long reads
sensing
3. High-Throughput Single-Cell Sequencing
 Allows sequencing of individual cells’ genomes or transcriptomes.
 Useful in developmental biology, cancer research, and immunology.
Future & Emerging Trends
 Nanopore improvements (higher accuracy, real-time sequencing)
 In situ sequencing (DNA/RNA sequencing in tissue samples)
 Integration with AI for base calling and variant detection
 Fourth-generation technologies — combining spatial, temporal, and chemical data
Summary Table
Generation Methods/Platforms Key Traits
1st Gen Sanger, Maxam-Gilbert Accurate, low throughput
2nd Gen (NGS) Illumina, Ion Torrent, Roche Massive parallel sequencing
 454
3rd Gen (TGS) PacBio, Oxford Nanopore Long reads, single-molecule
4th Gen AI-integrated, spatial, in situ Ultra-fast, real-time, multi-
(Emerging) layered