Emily Buckhouse

Emily Buckhouse
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
Determining the Sequence of DNA

Methods:
1. Chain termination or dideoxy method

F. Sanger
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
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
AGCATGCTGCAGTCATGCTTAGGCTA
Reconstruction
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 337340.
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 chainterminating 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.