Originally written by Emily Buckhouse

Why sequence DNA?

 Learn about all genes available for an
organism to use -- a very important tool for
biologists
 Not just sequence of genes, but also
positioning of genes and sequences of
Recombined DNA, takung DNA from one thing and
regulatory regions putting it in anotehr place

 Verify construction of recombinant DNAs

 Compare specific gene sequences among
species to estimate evolutionary
relationships
 And more….
History of DNA sequencing
History of DNA sequencing
The elegant idea
behind DNA sequencing
Fred Sanger
 Technology changes quickly, but for many
years we’ve used Sanger’s cool trick.
 In the 1970’s, Sanger’s group discovered a
fundamentally new method of 'reading' the
linear DNA sequence using special bases
called chain terminators or
dideoxynucleotides. Chain ends (Caboose)
 This method is still in use today.
 What is the basis of Sanger’s method?
Fascinating. Now how do we
actually sequence ??
 Dideoxy (Sanger) Method

Similar to
4 Steps:
replication with an enzyme = Helicase
and later: 1. Denaturation Or heat it up
PCR
2. Primer attachment and extension of bases
3. Termination New for sequencing

4. Gel electrophoresis
For DNA, seperate by size
Sanger dideoxy
sequencing--basic
method

3’ Single stranded DNA 5’

5’ 3’

a) Anneal the primer

DNA Polymerase Action
The phosphates give energy for the 5'
end to attach to the 3' OH

deoxyguanine
triphosphate
 Extendthe primer with DNA
polymerase in the presence of all four
dNTPs, with a limited amount of a
dideoxy NTP (ddNTP)
chain terminators (Missing and 3' OH)
Phosphodiester Bond
 DNA Polymerase

Terminator
Dideoxy (Sanger) Method
no hydroxyl on the 2' or 3' carbon

• ddNTP- 2’,3’-
dideoxynucleotide
• No 3’ hydroxyl
• Terminates chain
when incorporated
• Add enough so each
ddNTP is randomly
and completely
incorporated at each
base
Dideoxy Nucleotide
Sanger dideoxy
sequencing: basic
method
3’ T T T T 5’

Anywhere the is a T the chain could end

5’ 3’
ddA
T Binds to A ddATP in the
reaction: anywhere
ddA there’s a T in the
ddA
template strand,
occasionally a ddA
ddA will be added to the
growing strand
Dideoxies block elongation

ddGTP

H
X
DNA Sequencing Reactions
 Dideoxy
Method
• Run four separate
reactions each with
different ddNTPs
• Run on a gel in
four separate lanes
• Read the gel from
the bottom up

End in A

End in T

end in A
Determining the Sequence of DNA
 Methods:
1. Chain termination or dideoxy method
 F. Sanger
2. Shotgun sequence method
3. 2nd generation sequence methods
 Pyrosequencing
Overview: Dideoxy (Sanger) Method
2
3
Using temperature
(PCR)

1
4

Gel
electrophoresis
5
Automated Version of the Dideoxy
Method
So What’s Wrong With It?
 The dideoxy method is good only for
500-750bp reactions
 Expensive
 Takes a while
 The human genome is about 3 billion bp
Human Genome Project
 Began in 1990
 Why?
 Human evolution
 Nature versus nurture
 Causes of disease
Shotgun
Sequencing
 Used to sequence
whole genomes
 Steps:
 DNA is broken up
randomly into
smaller fragments
 Dideoxy method
produces reads
 Look for overlap of
reads

Strand Sequence
AGCATGCTGCAGTCATGCT-------
First Shotgun Sequence
-------------------TAGGCTA
AGCATG--------------------
Second Shotgun Sequence
------CTGCAGTCATGCTTAGGCTA
Reconstruction AGCATGCTGCAGTCATGCTTAGGCTA
2nd Generation: Pyrosequencing
 Sequencing by synthesis
 Advantages:
 Accurate
 Parallel processing
 Easily automated
 Eliminates the need for labeled primers and
nucleotides
 No need for gel electrophoresis
Pyrosequencing
 Basic idea:
 Visible light is generated and is proportional to the
number of incorporated nucleotides
 1pmol DNA = 6*1011 ATP = 6*109 photons at 560nm
DNA Polymerase I from E.coli.

pyrophospate

From fireflies, oxidizes luciferin and generates light

Pyrosequencing
 1st Method
 Solid Phase
○ Immobilized DNA
○ 3 enzymes
○ Wash step to remove nucleotides after each addition
Pyrosequencing
 2nd Method
 Liquid Phase
○ 3 enzymes + apyrase (nucleotide degradation enzyme)
 Eliminates need for washing step

• In the well of a microtiter

plate:
• primed DNA
template
• 4 enzymes
• Nucleotides are added
stepwise
• Nucleotide-degrading
enzyme degrade previous
nucleotides
Pyrosequencing Method:
Pyrosequencing Results:
Pyrosequencing Disadvantages
 Smaller sequences
 Nonlinear light response after more than
5-6 identical nucleotides
Summary
 DNA sequencing is a common procedure
 Dideoxy method
 Chain termination method
 Best for small DNA segments
 Whole genome shotgun sequencing
 Sequence human genome
 Fragments larger DNA strand to manageable
chunks
 Pyrosequencing
 Sequence by synthesis
 Accurate and fast
References
Applied Biosystems Automated DNA Sequence Chemistry Guide. (2000)

Garrett & Grisham. (2007) Biochemistry. Thomson and Brooks/Cole. 3rd ed. Pgs 337-
340.

Maxam, A. & Gilbert, W. (1977) A new method for sequencing DNA. Proc. Natl. Acad.
Sci. 74, 560-564.

Ronaghi, M. (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res.

11, 3-11.

Sanger, F., Nicklen, S., & Coulson, A.R. (1977) DNA Sequencing with chain-
terminating inhibitors. Proc. Natl. Acad. Sci. 94, 5463-5467.

Shendure, J. & Ji, H. (2008) Next-generation DNA Sequencing. Nature Biotech. 26,
1135-1145

Venter, C, et al. (2001) The sequence of the human genome. Science. 291, 1304.