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DNA Sequencing: Lecture - 3

This document provides an overview of DNA sequencing techniques. It discusses first generation Sanger sequencing from 1977, second generation techniques like 454/Roche from 2005 and Illumina from 2007 that provide shorter reads at lower cost. Third generation methods like Pacific Biosciences and Oxford Nanopore provide longer reads but with lower accuracy. Key terms like oligonucleotides, plasmids, and cloning vectors are also explained. The document aims to inform readers about the history and principles of DNA sequencing technologies.

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69 views

DNA Sequencing: Lecture - 3

This document provides an overview of DNA sequencing techniques. It discusses first generation Sanger sequencing from 1977, second generation techniques like 454/Roche from 2005 and Illumina from 2007 that provide shorter reads at lower cost. Third generation methods like Pacific Biosciences and Oxford Nanopore provide longer reads but with lower accuracy. Key terms like oligonucleotides, plasmids, and cloning vectors are also explained. The document aims to inform readers about the history and principles of DNA sequencing technologies.

Uploaded by

sk3 khan
Copyright
© © All Rights Reserved
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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DNA Sequencing

Lecture – 3

Nafis Neehal, Lecturer, Department of 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
1. DNA
Sequencing
Determining nucleotide sequences
DNA
Sequencing
▹DNA sequencing is the process of
determining the precise order of nucleotides
(A, T, G, C) within a DNA molecule.
2. First Generation Sequencing
Predominant method for sequencing for decades
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
Step 1 - DNA
Preperation

1 • Cut DNA into a smaller piece for


sequencing

• Insert into Plasmid


2 • Insert Plasmid inside Bacteria Cell and
let it multiply

• Extract all the necessary Plasmids and


3 from Plasmid, isolate the DNA for
sequencing

4
Step 2 – Sequencing
Reaction
▹ Strand Separation 1.
o
- Heat DNA in 96 C (denaturation)

▹ Primer Annealing
o
- Lower temperature to 50 C (annealing)
- Primer binds to DNA

▹ Primer Extension
o
- Increase temperature to 60 C 2.
- DNA Polymerase binds to Primer
- Add complimentary bases (dNTP) after Primer
until terminator base is added (ddNTP)
3
.
▹ Termination
- Terminate chain after ddNTP is added
- ddNTP is fluorescently labelled (different 4.
colors for A, T, G, C)
Step 3 – Electrophoresis in
▹
Capillary Sort the newly synthesized DNA strands
by length

▹ Strands are loaded inside a capillary tube

▹ An electrical negative charge pulls


positively charged DNA strands through
the capillary

▹ Emerged strands pass through a laser


beam that excites the ddNTP fluorescent
dye at the end of each strand

▹ Beam causes dye to glow in a specific


wavelength/color which is captured by
photocell and stored in a computer

▹ Computer than maps each color to each


nucleotide sequentially and generates
final sequence output
3. Second / Next Gen
Sequencing
Less Costly methods, mostly Short Read Sequences, High number of reads
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)
4. Third / Next-Next Gen
Sequencing
Long reads, Higher error rate
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)
5. Miscellaneous Terms
Some comparisons, terms etc.
Oligonucleotide
▹Short sequences of DNA or RNA

▹Typically less than 20bp

▹Oligonucleotide of ‘k’ bases length is called k-mer.


Denaturation and
Annealing
Denaturation
- Energy of heat pull apart two DNA
strands
- Happens at a critical temperature
denoted Tm

Annealing
- Decrease temperature, and strands are
joined back together
- Only complementary bases will bond
Plasmid
▹Small, circular piece of DNA often found in
bacteria.

▹Sizes of 2.5-20 kb

▹Plasmid using method -

* Isolate them in large quantities

* Cut and splice them, adding whatever DNA


needed

* Put them back into bacteria, where they'll


replicate along with the bacteria's own DNA

* Isolate them again - getting billions of copies


of whatever DNA was inserted into the plasmid
Bacterial Artificial Chromosome
(BAC)

▹Used like a plasmid

▹BACs carry DNA from humans or mice or any


other living being, and is inserted into a host
bacterium for replication

▹BAC is artificially constructed, unlike Plasmid


Cloning Vector
▹A cloning vector is a small piece of DNA, taken
from a virus, a plasmid, or the cell of a higher
organism, that can be stably maintained in an
organism, and into which a foreign DNA fragment
can be inserted for cloning purposes.
8%
Of Human DNA is made of Ancient Viruses

700 Terabytes
Data can be stored in 1gm DNA

50 Years Time
Type entire human genome at a speed of 60 wpm

99.9%
Human DNA is identical, 0.01% creates
human diversity
Impressed
TO BE
CONT
INUED

?
Youtube Links
▹Sanger Sequencing - https://www.youtube.com/watch?v=ONGdehkB8jU

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