dna sequencing: importance basic blueprint for life; aesthetics. gene and protein. –function...
TRANSCRIPT
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DNA sequencing: Importance• Basic blueprint for life; Aesthetics.• Gene and protein.
– Function– Structure– Evolution
• Genome-based diseases- “inborn errors of metabolism.”– Genetic disorders – Genetic predispositions to infection– Diagnostics– Therapies
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DNA sequencing methodologies
• Maxam-Gilbert – base modification by
general and specific chemicals.
– depurination or depyrimidination.
– single-strand excision.
– not amenable to automation
• Sanger– DNA replication.
– substitution of substrate with chain-terminator chemical.
– more efficient
– automation??
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Maxam-Gilbert chemical method
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versus “bio” based methods
• Sanger• dideoxynucleotides
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DNA biochemistry
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Sequence Masters• Fred Sanger, 1958
– Was originally a protein chemist
– Made his first mark in sequencing proteins
– Made his second mark in sequencing RNA
• 1980 dideoxy sequencing
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The Sanger Method• Random incorporation of a dideoxynucleoside
triphosphate into a growing strand of DNA• Requires DNA polymerase I.. Why?• Requires a cloning vector with initial primer
(M13, high yield bacteriophage, modified by adding: beta-galactosidase screening, polylinker)
• Uses 32P-deoxynucleoside triphosphates
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Sanger Method Sequencing Gel
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DNA sequencing: biochemistry
OC N
purineor
pyrimidine
P O
O
OH
P O
O
OH
P O
O
OH
HO
P O
O
OH
O OC N
purineor
pyrimidine
OH
5’
3’
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DNA sequencing: Sanger dideoxy method I
OC N
purineor
pyrimidine
P O
O
OH
P O
O
OH
P O
O
OH
HO
Hdideoxyribonucleoside triphosphate(ddNTP)
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DNA sequencing: Sanger II
OC N
purineor
pyrimidine
P O
O
OH
P O
O
OH
P O
O
OH
HO
P O
O
OH
O OC N
purineor
pyrimidine
H
chainterminationmethod
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DNA sequencing: Chemistry
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DNA sequencing: Chemistry
template + primers + polymerase +label at?
1dCTPdTTPdGTPdATP
ddATP*
2dCTPdTTPdGTPdATP
ddGTP*
3dCTPdTTPdGTPdATP
ddTTP*
4dCTPdTTPdGTPdATP
ddCTP*
extension
electrophoresis
A•TG•CA•TT•AC•GT•AG•CG•CA•TG•CT•AT•AC•GT•AG•CA•T
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Manual radioactive sequencing
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DNA sequencing: Chemistry
template + polymerase +
1dCTPdTTPdGTPdATP
ddATPprimer
2dCTPdTTPdGTPdATP
ddGTPprimer
3dCTPdTTPdGTPdATPddTTPprimer
4dCTPdTTPdGTPdATPddCTPprimer
extension
electrophoresis
A•TG•CA•TT•AC•GT•AG•CG•CA•TG•CT•AT•AC•GT•AG•CA•T
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Semi-automated fluorescent DNA sequencing
• Fred Sanger et. al., 1977.
• Walter Gilbert et. al., 1977.
• Leroy Hood et. al. 1986.
• Applied Biosystems, Inc.
• DuPont Company.
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DNA sequencing: upgrade, second iteration, terminator-label
• Disadvantages of primer-labels:– four reactions– tedious– limited to certain regions, custom oligos or– limited to cloned inserts behind ‘universal’
priming sites.
• Advantages:
• Solution:– fluorescent dye terminators
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DNA sequencing: Chemistrytemplate + polymerase +
dCTPdTTPdGTPdATP
ddATPddGTPddTTPddCTP
extension
electrophoresis
A•TG•CA•TT•AC•GT•AG•CG•CA•TG•CT•AT•AC•GT•AG•CA•T
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DNA sequencing: Chemistry
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Sequence Masters
• Walter Gilbert– Harvard physicist– Knew James Watson– Became intrigued with the
biological side– Became a biophysicist
• Allan Maxam
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The Maxam-Gilbert Technique
• Principle - Chemical Degradation of Purines– Purines (A, G) damaged by
dimethylsulfate– Methylation of base– Heat releases base– Alkali cleaves G– Dilute acid cleave A>G
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The 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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The Maxam-Gilbert Method
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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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Sequencing Gives:• The letters in a sentence• Remember Prions?
– Short sequence in genomes– Single nucleotide change in alleles
• Valine - Valine = not susceptible to BSE• Methionine - Valine = at risk• Methionine-methionine = watch out!
• How can we genetically screen for single nucleotide differences?
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Applications DNA sequencing
• Whole genome analysis
• Comparative genomics
• Applications to subfields