BIO282 Lecture Notes - Lecture 9: Dna Sequencing, Dideoxynucleotide, Chain Termination
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DNA SEQUENCING
Key points
• What are Primers, template DNA, deoxy and dideoxy- nucleotides?
• What is Sanger/chain termination method and how does it work?
• How does the automated dideoxy sequencing work?
• What are the Next Generation Sequencing Methods, how do they work?
• What are the Third Generation Sequencing Methods
DNA Sequencing
• DNA sequencing is a process that determines the precise order of bases in a piece of DNA.
• Major breakthrough came in 1977
• 1980 Nobel Prize: Sanger and Gilbert for DNA sequencing
• The Sanger method also known as a chain termination method
revolutionized DNA sequencing.
It was based on-
• Primer: a small oligonucleotide of a known sequence
• DNA polymerase: an enzyme that synthesizes DNA
• Dideoxynucleotides: to terminate DNA synthesis
DNA polymerase
In a presence of a template DNA, a primer and deoxynucleotides, DNA polymerase synthesizes
DNA by adding complementary bases at the end of the primer/growing chain.
Will add the next one but it can’t differentiate. The different colour show the different
things are added. Remember there are thousands of molecules available. At each
place there is an equal opportunity for them to be added. Either it can incorporate a
terminator or not.
DNA template
5`-ATGCGTGCGTACATACGCAATTCGAACTGCGCTAACGGCCTTGCG-3`
Primer
5`-CGCAAGGCCGTTAGCGCAG-3`
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• No OH group on the sugar
• If a ddNTP incorporates into a growing chain, it causes termination of DNA synthesis. Why?
o There is no OH group to form a phosphodiester bond between the 3' and 5' hydroxyl
Reactions are performed in 4 tubes each containing a template, a primer, all four dNTPs and a single
ddNTP
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• This method allows you to have different sized fragments