DNA Sequencing
Nitrogenous Bases
Nucleosides
⦿ Base linked to a 2-deoxy-D-ribose at 1’ carbon
Nucleotides
• Nucleosides with a phosphate at 5’ carbon
Phosphodiester Bond
⦿ DNA Polymerase
⦿ Methods:
Determining 1. Chain termination
or dideoxy
the Sequence method
⚫ F. Sanger
of DNA 2. Shotgun
sequence method
3. 2nd generation
sequence
methods
⚫ Pyrosequencing
Dideoxy (Sanger) Method
⦿ 4 Steps:
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Gel electrophoresis
Overview: Dideoxy (Sanger) Method
2
3
1
4
Gel
electrophoresis
5
Dideoxy (Sanger) Method
• 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
Method
• Run four separate
reactions each with
different ddNTPs
• Run on a gel in
four separate lanes
• Read the gel from
the bottom up
Automated Version of the Dideoxy
Method
⦿ The dideoxy
So What’s method is good
only for 500-750bp
Wrong With reactions
It? ⦿ Expensive
⦿ Takes a while
⦿ The human
genome is about 3
billion bp
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 [Link].
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 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.
QUESTIONS?