Download DNA Sequencing

DNA Sequencing
BCH 446
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DNA sequencing
 Determination of nucleotide sequence
 the determination of the precise sequence of nucleotides in a
sample of DNA
 Two similar methods:
1. Maxam and Gilbert method
2. Sanger method
 They depend on the production of a mixture of oligonucleotides labeled
either radioactively or fluorescein, with one common end and differing in
length by a single nucleotide at the other end
 This mixture of oligonucleotides is separated by high resolution
electrophoresis on polyacrilamide gels and the position of the bands
determined
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The Maxam-Gilbert
Technique
• Principle - Chemical Degradation
of Purines
– Purines (A, G) damaged by
dimethylsulfate
– Formic acid modifies G &A
– Methylation of base
– Heat releases base
– Alkali cleaves G
– Dilute acid cleave A>G
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Maxam-Gilbert
Technique
• Principle- Chemical
Degradation of
Pyrimidines
– Pyrimidines (C, T) are
damaged by hydrazine
– Piperidine cleaves the
backbone
– 2 M NaCl inhibits the
reaction with T
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Maxam and Gilbert Method
Chemical degradation of purified fragments (chemical degradation)
The single stranded DNA fragment to be sequenced is end-labeled by
treatment with alkaline phosphatase to remove the 5’phosphate
It is then followed by reaction with P-labeled ATP in the presence of
polynucleotide kinase, which attaches P labeled to the 5’terminal
The labeled DNA fragment is then divided into four aliquots, each of which is
treated with a reagent which modifies a specific base
1. Aliquot A + dimethyl sulphate, which methylates guanine residue
2. Aliquot B + formic acid, which modifies adenine and guanine residues
3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues
4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine
The four are incubated with piperidine which cleaves the sugar phosphate
backbone of DNA next to the residue that has been modified
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Maxam-Gilbert
sequencing - modifications
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Maxam-Gilbert sequencing - summary
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Advantages/disadvantages
Maxam-Gilbert sequencing
 Requires lots of purified DNA, and many intermediate purification steps
 Relatively short readings
 Automation not available (sequencers)
 Remaining use for ‘footprinting’ (partial protection against DNA
modification when proteins bind to specific regions, and that produce
‘holes’ in the sequence ladder)
In contrast, the Sanger sequencing methodology requires little if
any DNA purification, no restriction digests, and no labeling of
the DNA sequencing template
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Original Sanger Method
 Random incorporation of a dideoxynucleoside
triphosphate into a growing strand of DNA
 Requires DNA polymerase I
 Requires a cloning vector with initial primer (M13, high
yield bacteriophage, modified by adding: betagalactosidase screening, polylinker)
 Uses 32P-deoxynucleoside triphosphates
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Sanger Method
 in-vitro DNA synthesis using ‘terminators’, use of dideoxinucleotides that do not permit chain elongation after their
integration
 DNA synthesis using deoxy- and dideoxynucleotides that
results in termination of synthesis at specific nucleotides
 Requires a primer, DNA polymerase, a template, a mixture of
nucleotides, and detection system
 Incorporation of di-deoxynucleotides into growing strand
terminates synthesis
 Synthesized strand sizes are determined for each dideoxynucleotide by using gel or capillary electrophoresis
 Enzymatic methods
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Dideoxynucleotide
PPP
O
5’
CH2
O
BASE
3’
no hydroxyl group at 3’ end
prevents strand extension
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The principles
• Partial copies of DNA fragments made with DNA
polymerase
• Collection of DNA fragments that terminate with
A,C,G or T using ddNTP
• Separate by gel electrophoresis
• Read DNA sequence
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3’
primer 5’
CCGTAC 5’
3’
dNTP
ddATP ddTTP
GGCA
ddCTP ddGTP
GGCAT
A
T C
GGC
G
G
GG
GGCATG
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Comparison
• Sanger Method
– Enzymatic
– Requires DNA synthesis
– Termination of chain
elongation
• Maxam Gilbert Method
– Chemical
– Requires DNA
– Requires long stretches of
DNA
– Breaks DNA at different
nucleotides
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