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

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??

Maxam-Gilbert chemical method

versus “bio” based methods

• Sanger• dideoxynucleotides

DNA biochemistry

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

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

Sanger Method Sequencing Gel

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’

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)

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

DNA sequencing: Chemistry

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

Manual radioactive sequencing

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

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.

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

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

DNA sequencing: Chemistry

Sequence Masters

• Walter Gilbert– Harvard physicist– Knew James Watson– Became intrigued with the

biological side– Became a biophysicist

• Allan Maxam

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

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

The Maxam-Gilbert Method

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

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?

Applications DNA sequencing

• Whole genome analysis

• Comparative genomics

• Applications to subfields