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DNA Sequencing

ukzn rdna 202(Lecture 16)Lecture 16

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13 views24 pages

DNA Sequencing

ukzn rdna 202(Lecture 16)Lecture 16

Uploaded by

lindokuhlenkosiyethu90
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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Next Section:

DNA Sequencing

RDNA202

Cassie
[email protected]
Lectures
Lecture 16: DNA Sequencing Theory & Sanger Sequencing

Lecture 17 & 18: DNA Sequencing Theory & NGS


Sanger
Sequencing

RDNA202

Cassie
[email protected]

https://www.cbehx.co.uk/product/next-generation-sequencing-ngs/
DNA Sequencing
•The process of determining the order of bases in a length of
DNA
• Has helped advance our understanding of genetics.
Applications of DNA Sequencing
• Tackling Human Disease
• Identification of mutations associated with diseases – e.g. cancer

• Identifying variations and polymorphisms


• In the human genome
• Provides information about Single Nucleotide Polymorphisms (SNPs)

• Studying evolution:
• How genes and genomes evolve
Sanger Sequencing method
• Also known as “chain termination method”

• Classified as a First Generation Sequencing method

• Used to determine the nucleotide sequence of DNA

• Developed in 1977
• by two time Nobel Laureate – Frederick Sanger and his colleagues
• Father of genomics: 13 August 1918 – 19 November 2013
• Fred and colleagues – first scientists to sequence whole genome
• Genome was just over 5000 bases – from virus called phiX174 – infects bacteria
Sanger Sequencing method

• Uses high fidelity DNA-dependent polymerase

• Create a complementary copy to a single stranded DNA template

• Single primer – complementary to template


• Initiates DNA synthesis from 3’ end.
Sanger Sequencing – Chain Termination Method
Sanger Sequencing - ddNTPs
• Resemble DNA monomers
• Incorporated into growing chain

• Differ from dNTPs in two ways:


1. Lack 3’ hydroxyl group required for further DNA extension
• Leads to chain termination once incorporated

2. Each ddNTP has fluorescent dye attached


• Allows for automatic detection
Nucleotides: Deoxytriphosphate Vs Dideoxytriphosphate
Components Required for Sanger Sequencing

Note: Only ONE Primer


is used
Sanger Sequencing Method – Step 1
1. The DNA to be sequenced must first be amplified by PCR
• Then DNA double helix is denatured with heat
• Separates the two strands
• These will be the templates for DNA synthesis
Sanger Sequencing Method – Step 2
2. Generation of n DNA fragments of varying lengths
• Each terminated with a fluorescently labelled nucleotide

• Where n = the number of nucleotide bases in the target DNA sequence

• This is done by combining:


• DNA primer
• Nucleotides – deoxynucleotide triphosphates (dNTPs =dATP, dTTP, dCTP, dGTP)
• Labelled Dideoxynucleotides (ddNTPs = ddATP, ddTTP, ddCTP, ddGTP)
Sanger Sequencing Method – Step 2 continued
2. Labelled Dideoxynucleotides (ddNTPs =
ddATP, ddTTP, ddCTP, ddGTP)

• No nucleotide can be added to the chain once


a ddNTP has been added

• Each fragment will end with a labelled


nucleotide

• Therefore – much smaller amount of ddNTPs


are used compared to the normal dNTPs
Sanger Sequencing Method: Chain Termination

GROWING DNA CHAIN TERMINATES DUE TO


INCORPORATION OF A DDNTP.
Sanger Sequencing Method – Step 3
3. Capillary electrophoresis
• Separate amplicons of various lengths

• These amplicons – terminated with ddNTP at 3’ end

• Principles of electrophoresis:
• Shorter fragments move faster than longer fragments

• Results in DNA being analysed from shortest to longest


sequence
Sanger Sequencing Method – Capillary electrophoresis
• Shorter fragments move faster than longer fragments
Sanger Sequencing
Example: Reading
Results
Sanger Sequencing Method:
The process
The DNA to be sequenced is prepared as a single strand.
This template DNA is supplied with a mixture of all four
normal (deoxy) nucleotides in ample quantities
dATP
dGTP
dCTP
dTTP

a mixture of all four dideoxynucleotides, each present in


limiting quantities and each labeled with a "tag" that
fluoresces a different color:
ddATP
ddGTP
ddCTP
ddTTP, and
DNA polymerase I
Sanger Sequencing Method – Step 4
4. In the final step:
• Laser excites label on nucleotide at the end of each sequence
• Each base is tagged with a different label
• Light emitted by each excited nucleotide – tied to correct base
• Laser generates a chromatogram
• Shows fluorescent peak of each nucleotide
• Nucleotides are in correct order because of electrophoresis.
Sanger Sequencing Method – Step 4
Sanger Sequencing Method – Step 4
Sanger Sequencing Overview
DNA Sequencing:
Next Generation
Sequencing (NGS)

RDNA202

Cassie
[email protected]

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