Chemical Synthesis, Amplification, and Sequencing of DNA (Part I)
DNA Sequencing - Assiut University sequencing.pdfThe Sanger DNA sequencing method - Uses dideoxy...
Transcript of DNA Sequencing - Assiut University sequencing.pdfThe Sanger DNA sequencing method - Uses dideoxy...
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DNA Sequencing
Dr. Serageldeen A. A. Sultan PhD in Molecular virology
Yamaguchi University, Japan (2010)
Lecturer of virology
Dept. of Microbiology
SVU, Qena, Egypt
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-Determining the precise order of nucleotides in a
piece of DNA
-DNA sequence is useful in studying fundamental
biological processes and in applied fields such as
diagnostic or forensic research
-DNA sequencing methods have been around for
40 years, and since the mid-1970s
What is DNA sequencing?
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Nobel Prize in Chemistry in 1980
Founders of sequencing technology
Sanger Gilbert
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Base
Sugar
Acid
Phosphate
Adenine
Guanine
Thymine
Cytosine
Uracil
Nucleoside
Nucleotide
Purine
Pyrimidine
Ribose, Deoxyribose
1’
2’ 3’
4’
5’
Basic structure of nucleic acid
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P
R
B
3’
2’
5’
1’
A T C G A T C G
P OH
5’ 3’
5’ pApTpCpGpApTpCpG-OH 3’
5’ pATCGATCG-OH 3’
ATCGATCG
Nucleotides linked by phosphodiester
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5’ pCpGpApTpCpGpApT-OH 3’ 5’ pApTpCpGpApTpCpG-OH 3’
5’ 3’
3’ 5’
Two strands are antiparallel
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Two basic methods for DNA sequencing :-
A- Chemical cleavage method (Maxam and Gilbert, 1977)
- Base-specific cleavage of DNA by certain chemicals
- Four different chemicals, one for each base
- A set of DNA fragments of different sizes
- DNA fragments contain up to 500 nucleotides
B- Enzymatic method (Sanger, 1981)
Sequencing methods
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- Dimethyl sulfate
(DMS) mehylates G
-Acid (A)
-Hydrazine (C)
- Hydrazine & NaCl
(T)
- Piperidine
The chain cleavage reaction
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32pGpCpTpGpCpTpApGpGpTpGpCpCpGpApGpC
G G G G G G G
32p 32pGpCpTp 32pGpCpTpGpCpTpAp 32pGpCpTpGpCpTpApGp 32pGpCpTpGpCpTpApGpGpTp 32pGpCpTpGpCpTpApGpGpTpGpCpCp 32pGpCpTpGpCpTpApGpGpTpGpCpCpGpAp 32pGpCpTpGpCpTpApGpGpTpGpCpCpGpApGpC
The fragments created by chain cleavage at guanines
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Chemical degradation method (Maxam–Gilbert method)
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The Sanger DNA sequencing method
- Uses dideoxy nucleotides to terminate DNA synthesis.
- DNA synthesis reactions in four separate tubes
- Radioactive dATP is also included in all the tubes so the
DNA products will be radioactive.
-Yielding a series of DNA fragments whose sizes can be
measured by electrophoresis.
- The last base in each of these fragments is known.
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O
H H
O = P – O
O
O
2’, 3’ dideoxy nucleotide
Can not form phosphodiester
bound with next coming dNTP
Base
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The dideoxy sequencing method (Sanger method)
A labeled primer is used
to initiate DNA synthesis.
The addition of four
different dideoxy
nucleotides randomly
arrests synthesis.
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The resulting fragments are
separated electrophoretically
and subjected to autoradiography
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Recent methods of chain termination sequencing
Thermal cycler sequencing
Automated DNA sequencing
Pyrosequencing
Sequencing by hybridization
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-The primer extension reactions are run in the same
way as in the manual method
-Reaction carried out in one tube and all possible
products are actually produced
- The various reaction products separate according to
size on gel electrophoresis
Automated DNA sequencing
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-The bands are color-coded according to the termination
reaction that produced them
-A laser scanner excites the fluorescent tag on each band as
it passes by, and a detector analyzes the color of the
resulting emitted light
- Each colored peak is a plot of the fluorescence intensity
of a band as it passes through the laser beam
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Printout of an automated DNA sequencing
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Noisy or weak signal
- Partially failed sequencing reaction.
- Too much or too little DNA.
- Partial loss of sample during clean-up
secondary peaks
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Mixed DNA templates
- Two or more templates were present in the reaction
- Two primers were present in the sequencing reactions
- Two priming sites are present in DNA template
- Different sequencing reactions were accidentally mixed at the clean up stage
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Short DNA sequencing read lengths
- Too much template DNA
- Excessive dilution of the BigDye reagent
- Too much primer
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Blurry trace chromatogram peaks
- Capillary overload (dirty samples with large amount of DNA, proteins or salts)
- High sequencing run voltages
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Minor dye blob Moderate dye blob
-Poor post-sequencing clean up
-Lost pellet
-Failed reaction
Dye Blobs (excess free dye terminators)
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Sharp trace signal spikes
Small gas bubbles forming in the capillaries during electrophoreses
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Sequence Slippage
Long runs of a mono nucleotide base
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mixed sequencing after the seven T
Small G peak preceding the large single G peak
Template insertion and deletions
- Direct sequencing of PCR products derived from polymorphic templates
- Random mutation has occurred during the cloning process
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Chimeras and sequence rearrangements
- Cloning two or more unrelated DNA fragments at once in the same vector
- 'Unstable' sequences like repeats as long poly A tails
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Delayed sequencing signal start
- Capillary overload
The two most common causes of capillary overload are:-
too much template DNA
dirty template DNA contaminated with proteins an/or salt
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Late "G" dye peaks
The breakdown of the BigDye sequencing chemistry before sample clean up
(excess heating or high numbers of freeze/ thaw cycles)
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What can sequence tell us?
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-Knowledge about the physical nature of genome
- Classification of microorganisms
- Relationship among diverse genomes
- Information about
- the origin of microorganism
-its movement in different population
-Searching for restriction endonuclease cleavage sites
- Vectors designed to over-express proteins or to
generate protein
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-Identification of protein-coding regions (ORF)
within the DNA sequence
Similarity of DNA and protein data bases, can
lead to important insights about the function
and structure of a cloned gene and its product
-Analysis of the 5′ and 3′ non-coding regulatory
regions of a gene.
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-DNA sequence information is essential for site
directed mutagenesis
-DNA sequence information (sequence tagged
sites, or STS; or expressed sequence tags, or
ESTs) are the basis of methods for mapping and
ordering large DNA segments cloned into yeast
or bacterial artificial chromosomes (CYACs;
BACs) or cosmids
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Thank you