DNA Sequencing
Prepared By
Fatema Tuj Johora
Assistant Professor, CSE,DIU
Contents
1. DNA Sequencing
2. First Gen Sequencing
-Sanger Method (1977)
3. Second / Next Gen Sequencing
- 454/Roche (2005)
- ABI SOLiD (2006)
- Illumina/Solexa (2007)
4. Third / Next-Next Gen Sequencing
- Pacific Biosciences (PacBio)
- Oxford Nanopore
5. Miscellaneous Terms
DNA Sequencing
► DNA sequencing is the process of determining the precise order
of nucleotides (A, T, G, C) within a DNA molecule.
Miscellaneous Terms
► An oligonucleotide is a short strand of nucleotides (the
building blocks of DNA or RNA) that is typically made up of
fewer than 50 nucleotides. Oligonucleotides can be
synthesized artificially and are used in a wide range of
molecular biology applications, including DNA sequencing,
PCR (polymerase chain reaction), gene synthesis, and
other research or diagnostic purposes.
Cloning vector
► A cloning vector is a DNA molecule used to carry foreign
DNA into a host cell for the purpose of cloning or
amplifying that DNA. Cloning vectors are specially
designed to facilitate the insertion, replication, and
sometimes expression of foreign genes or DNA fragments
within a host organism, usually bacteria (like E. coli),
yeast, or mammalian cells.
Plasmid
► In DNA sequencing, plasmids are commonly used
as cloning vectors to insert and carry the DNA
fragment of interest to be sequenced. These
plasmids can then be used in a sequencing reaction
to determine the nucleotide sequence of the
inserted gene or DNA fragment.
Bacterial Artificial Chromosome
(BAC)
► A Bacterial Artificial Chromosome (BAC) is a type of
cloning vector that is specifically designed to carry very
large fragments of DNA (typically 100 to 300 kilobases, or
kb) in bacterial cells. BACs are used primarily for cloning
large pieces of DNA, such as entire genes, gene clusters, or
genomic libraries. They are an essential tool in genomics,
especially in large-scale projects like the Human Genome
Project.
First Generation Sequencing: Sanger Method
▹Developed by Frederick Sanger in 1977
▹Most popular and predominant method for DNA
Sequencing for decades
▹Can read up to 2000 bps
▹Slow and expensive
▹Labor intensive
▹Human Genome Project was completed using
Sanger Sequencing
Sanger Method
Step 1 - DNA Preparation
Step 2 –Sequencing Reaction
Step 3 – Electrophoresis
Step-1 DNA Preparation:
1. Cut DNA into a smaller piece for sequencing 1
2. Insert into Plasmid
2
3. Insert Plasmid inside Bacteria Cell and let it multiply
4. Extract all the necessary Plasmids and isolate the DNA 3
for sequencing
4
Step-2 Sequencing Reaction
1. Strand Separation (Denaturation)
► Heat the DNA to 96°C: The DNA sample is heated to a high
temperature (typically 95-98°C) to break the hydrogen bonds between
the two strands of the DNA, separating them into single-stranded
molecules. This process is known as denaturation.
Step-2 Sequencing Reaction
2. Primer Annealing
∙ After heating the DNA to separate its strands, the temperature is
lowered to about 50°C. This cooler temperature lets the primer
to bind the matching parts of the single DNA strands. This step is
called annealing.
Step-2 Sequencing Reaction
► Step 1:
Step 2:
Primer Extension
∙ Step 3: Incorporation of complementary bases: The DNA
polymerase adds complementary nucleotides (dNTPs) —
deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP) — one by
one, in the 5' to 3' direction, based on the sequence of the
template DNA.
►
Step 2: Termination
∙ Step 4: Termination of DNA strand:
∙ After production special building block called ddNTP is added instead of a normal dNTP.
∙ ddNTPs are missing a tiny part (the 3' –OH group) needed to add more bases.
∙ When a ddNTP is added, the DNA strand stops growing because no more bases can be
attached.
There are four types of ddNTPs (ddATP, ddTTP, ddCTP, ddGTP), each with a different
colored tag
(fluorescent dye).
∙ This color helps scientists know which base was added at the end of the stopped DNA
strand.
Step 3 – Electrophoresis in
Capillary
► The pieces of different lengths are separated, usually by a machine that reads the colors.
► Strands are loaded inside a capillary tube and sort the DNA strands by length .
► The capillary tube has electrodes (metal wires) at both ends connected to a power supply.
► When DNA is placed in the tube, the electric field causes the DNA molecules to move through the tube
toward the positive electrode.
► Emerged strands pass through a laser beam that excites the ddNTP fluorescent dye at the end of each strand
► Computer than maps each color to each nucleotide sequentially and generates final sequence output
Step 3 – Electrophoresis in
Capillary
Second / Next Gen Sequencing:
454/Roche (2005)
• Pyrosequencing technique
• Long Read Sequencing (length up to 700 bps)
• Accuracy 99.9%
• Can sequence up to 1 Million reads/run
• Fast (around 24 hours/run)
• Expensive (costs around $10 per 1 million base)
ABI SOLiD (2006)
• SOLiD (Sequence by Ligation)
• Short Read Sequencing (length up to 100 bps)
• Accuracy 99.9%
• Can sequence up to 1.4 Billion reads/run
• Time around 1-2 weeks, Slower than other sequencers
• Cheap (costs around $0.13 per 1 million base)
Illumina / Solexa (2007)
• Sequencing by Synthesis
• Short Read Sequencing (length up to 300 bps)
• Accuracy 99.9%
• Can sequence up to 3 Billion reads/run
• Moderately Slow (around 1-11 days/run)
• Expensive Equipment, run cost is low (costs around $0.05-
$0.15)
Third / Next-Next Gen Sequencing
Pacific Biosciences (PacBio)
• Single Molecule Real Time Sequencing
• Long Read Sequencing (length up to 40,000 bps)
• Accuracy 87%
• Can sequence up to 500-1000 Mega reads/run
• Time around 30 minutes – 4 hours, Faster
• Expensive Equipment, run cost is low (costs around $0.13-
$0.60)
Oxford Nanopore
• Nanopore sequencing
• Very Long Read Sequencing (length up 500 kb), Portable
• Accuracy 92-97%
• Depends on read length selected by user
• Time around 1 minutes – 48 hours, Faster
• Expensive Equipment, run cost is low (costs around $500-
$999 per flow cell)