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DNA Sequencing

DNA Sequencing Methods

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Hager El mahashhash
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25 views23 pages

DNA Sequencing

DNA Sequencing Methods

Uploaded by

Hager El mahashhash
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 PPT, PDF, TXT or read online on Scribd
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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
Dideox
y
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
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.

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