DNA Sequencing

Lecture 7

©2001 Timothy G. Standish

 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
 Sequenced Genomes
Currently the sequencing of at least 5 multicelled
eukaryotic genomes has been completed:
1998 Caenorhabditis elegans - 8 x 107 bp - A nematode
worm
2000 Homo sapiens - 3 x 109 bp - Humans
2000 Arabidopsis thaliana - 1.15 x 108 - A plant related to
mustard
2000 Drosophila melanogaster - 1.65 x 108 bp - Fruit flies
2002 Anopheles gambiae – 2.78 x 108 bp mosquito vector
of malaria

©2001 Timothy G. Standish

 New Technology
Rapid sequencing of large complex genomes
has been made possible by:
Foundational work done over many years
and…
Dramatic improvement in DNA sequencing
technology over the past few years

©2001 Timothy G. Standish

 Basic Principles
All current practical DNA sequencing techniques
can be divided into four major steps:
1. Labeling of DNA so that small quantities can be
easily detected, traditionally done by labeling with
either P32 or S35
2. Generation of fragments for which the specific
bases at the 3’ end are known
3. Separation of fragments using gel electrophoresis
sensitive enough to resolve differences in size of
one nucleotide
4. Fragment detection
©2001 Timothy G. Standish
 Maxam-Gilbert

• Walter Gilbert
– Harvard physicist
– Knew James Watson
– Became intrigued with
the biological side
– Became a biophysicist
• Allan Maxam
 The Maxam-Gilbert
Chemical Method
Three major steps:
1. DNA to be sequenced is typically labeled at the
5’ end using P32
2. Fragments are generated using chemicals that
break DNA at specific bases
3. These fragments are then separated and detected
using autoradiography
Polyacylamide Gel Electrophoresis is typically
used to separate fragments on the basis of single
nucleotide differences ©2001 Timothy G. Standish
 2 Fragment Generation
A number of chemicals will specifically
modify the bases in DNA
Modified bases can then be removed
from the deoxyribose sugar to which
they are attached on the sugar-phosphate
DNA backbone
Piperidine, a volatile secondary amine, is
used to cleave the sugar-phosphate
backbone of DNA at sites where bases
were modified ©2001 Timothy G. Standish
 Cleavage at Specific Bases
Typically 5 reactions are run:
1. Dimethylsulfate at pH 8.0 results in modification of
guanine (G)
2. Piperidine formate at pH 2.0 breaks glycosidic bonds
between deoxyribose and both purines, guanine (G)
and adenine (A), by protonation of nitrogen atoms
3. Hydrazine (rocket fuel!) opens pyrimidine rings on
both pyrimidines, cytosine (C) and thymine (T)
4. Hydrazine in the presence of 1.5 M NaCl only reacts
with C
5. 1.2 N NaOH at 90 oC strongly cleaves at A and may
also weakly cleave at C
©2001 Timothy G. Standish
 Cleavage at Specific Bases
The trick in chemical sequencing is to not allow
the reactions to go to completion
Partial reactions run using the following
conditions will result in a series of labeled DNA
fragments whose final base is known:
Dimethylsulfate at pH 8.0 -----------> G
Piperidine formate at pH 2.0 -------> G and A
Hydrazine ------------------------------> C and T
Hydrazine in 1.5 M NaCl -----------> C
1.2 N NaOH at 90 oC -----------------> A and some C
©2001 Timothy G. Standish
 32
Partial Reactions:
P Dimethylsulphate pH 8.0
5’*NNGACGTACTTA3’
5’*NNGACGTACTTA3’
5’*NNGACGTACTTA3’
5’*NNGACGTACTTA3’
5’*NNGACGTACTTA3’
5’*NNGACGTACTTA3’
5’*NNGACGTACTTA3’
©2001 Timothy G. Standish
 Partial Reactions:
Dimethylsulphate pH 8.0
5’*NNGACGTACTTA3’
Modification of
5’*NNGACGTACTTA3’ some, but not all,
5’*NNGACGTACTTA3’ of the G bases as
5’*NNGACGTACTTA3’ the reaction is not
allowed to go to
5’*NNGACGTACTTA3’
completion
5’*NNGACGTACTTA3’
5’*NNGACGTACTTA3’
©2001 Timothy G. Standish
 Partial Reactions:
Dimethylsulphate pH 8.0
Following breaking of the DNA strand at positions where G
was chemically modified, two sets of fragments result: 1) A
labeled set all ending where a G once was and 2) An unlabeled
set which cannot be detected using autoradiography
5’*NN3’ 5’ACGTACTTA3’
Labeled
5’*NNGAC 3’ fragments all of 5’TACTTA3’
which represent
5’*NNGAC 3’ a place where 5’TACTTA3’
G used to be
5’*NN3’ 5’ACGTACTTA3’
Unlabeled
5’*NN3’ fragments
5’TACTTA3’
5’*NN3’ undetectable using5’
autoradiography ACGTACTTA3’
©2001 Timothy G. Standish
 Partial Reactions:
Hydrazine
5’*NNGACGTACTTA3’
Some, but not
5’*NNGACGTACTTA3’ all, C and T
5’*NNGACGTACTTA3’
bases are
5’*NNGACGTACTTA3’
modified as the
5’*NNGACGTACTTA3’
5’*NNGACGTACTTA3’
reaction is not
5’*NNGACGTACTTA3’
allowed to go
to completion
©2001 Timothy G. Standish
 Partial Reactions:
Hydrazine
Following breaking of the DNA strand at positions where C or
T was chemically modified, two sets of fragments result: 1) A
labeled set all ending where a C or T once was and 2) An
unlabeled set which cannot be detected using autoradiography
5’*NNGA3’ 5’G3’ 5’ACTTA3’
Labeled
5’* NNGACG 3’
T C set
5’ACTTA3’
5’*NNGACGTAC3’ Unlabeled
fragments
5’ *A3’
5’*NNGA3’ 5’GTACTTA3’
5’*NNGACGTACT3’ *A3’
5’
5’*NNGACGTAC3’ 5’*TA3’
©2001 Timothy G. Standish
 Separation of DNA Fragments
All current practical sequencing
methods rely on separation of DNA
fragments in such a way that
differences in length of a single base
can be resolved
This is typically done using
polyacrylamide gel electrophoresis

©2001 Timothy G. Standish

 Separation of Fragments:
Maxam-Gilbert

3’
X

X
Dimethyl Piperidine Hydrazine Hydrazine 1.2 N
sulfate formate in 1.5 M NaOH at
pH 8 pH 2 NaCl 90 oC
G G+A T+C C A>C
to
X

5’GACGTACTTA3’
5’
X

G G+A T+C C A>C

©2001 Timothy G. Standish
 Disadvantages
Toxic chemicals
Large amounts of radioactivity
Sometimes ambiguous and
frequently ugly sequencing gels
Tricky to read autorads
Lack of automated methods
©2001 Timothy G. Standish
 Sanger Method
 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
 Sanger Sequencing
The Sanger sequencing method takes advantage of
the way that normal DNA replication occurs
For DNA to be extended using normal DNA
polymerases, a hydroxyl group must be present at
the 3’ carbon on deoxyribose
Fragments are generated by spiking reactions with
small quantities 2’ 3’ dideoxy nucleotides which
terminate polymerization whenever they are
incorporated into DNA
Polymerases used must lack 3’ to 5’ exonuclease
proofreading activity for this method to work
©2001 Timothy G. Standish
 Dideoxynucleotides
DNA Sequencing using the 2’-dideoxynucleotide
Sanger method involves the use monophosphate
of 2’3’-dideoxynucleotide Phosphate OH
triphosphates in addition to HO P O NH2
Base
regular 2’-deoxynucleotide O
N N
triphosphates N N
5’CH 2
Because 2’3’-dideoxynucleotide O
4’ 1’
triphosphates lack a 3’ hydroxyl 3’ Sugar 2’
group, and DNA polymerization
H
OH H
occurs only in the 3’ direction,
once 2’3’-dideoxynucleotide
triphosphates are incorporated, 2’3’-dideoxynucleotide
primer extension stops monophosphate
©2001 Timothy G. Standish
 OH
H OH
NH2
HO P O
N
O N
N O
CH2 N CH2

2’3’
O
O

dideoxy- O H
O P

OH
H
OH

O
nucleotides HO P

O
O
N
NH
H

Terminate CH2
O
N N NH2 O
CH2

DNA O

Replication
O
P HO
O H
NH2 H O
H H2O
HO P O
N
O
N O
CH2
O
O CH2
O

O HO
OH H P
HO
 Making DNA Fragments
In Sanger DNA sequencing reactions all the basic
components needed to replicate DNA are used
4 reactions are set up, each containing:
– DNA Polymerase
– Primer
– Template to be sequenced
– dNTPs
– A small amount of one ddNTP
ddATP, ddCTP, ddGTP, ddTTP
As incorporation of ddNTPs terminates DNA replication,
a series of fragments is produced all terminating with the
ddNTP that was added to each reaction
©2001 Timothy G. Standish
 DNA Sequencing
Cloned
fragment

Primer
Primer Binding sites

Plasmid (or phage)

with cloned DNA
fragment

©2001 Timothy G. Standish

 The ddATP Reaction
3’AATAGCATGGTACTGATCTTACGCTAT5’
5’TTATCG
5’TTATCGTACCATGACTAGATGCGATA
5’TTATCGTACCATGACTAGA
5’TTATCGTACCATGA
5’TTATCGTA

5’TTATCGTA
5’TTATCGTACCA
5’TTATCGTACCATGA
5’TTATCGTACCATGACTA
5’TTATCGTACCATGACTAGA
5’TTATCGTACCATGACTAGATGCGA
5’TTATCGTACCATGACTAGATGCGATA

©2001 Timothy G. Standish

Separation of Sanger Fragments
Products from 4 reactions ddATP ddCTP ddGTP ddTTP

each containing a small

amount of a
dideoxynucleotide are

Read 5’ to 3’ from bottom to top

loaded onto a gel
Because polymerization
goes 5’ to 3’ shortest
fragments are 5’ compared
to longer fragments which
are in the 3’ direction

©2001 Timothy G. Standish

A C G T DNA Sequencing
What A Sequencing
Autorad Actually
Looks Like

To read the autorad it is important to

start at the bottom and work up so that it
is read in the 5’ to 3’ direction

5’CTAGAGGATCCCCGGGTACCGAGCT...3’

©2001 Timothy G. Standish

Sequencing Method Refinements
Because of difficulties intrinsic to the Maxam-
Gilbert chemical sequencing strategy, efforts at
improvement have been concentrated on the Sanger
method
Major improvements in the following areas have
been achieved
Labeling and detection
Fragment separation
DNA Polymerases used in sequencing and resulting
strategies for generation of fragments
Automation ©2001 Timothy G. Standish
 Pros and Cons of the
Sanger Method
It is more amenable to automation than Maxam-
Gilbert
Fewer dangerous chemicals are used, but
acrylamide and P32 or S35 are still a problem
Gels or autorads are generally cleaner looking and
the reading of bases is a lot easier than Maxam-
Gilbert data
The bottom line: Without improvements in
automation, detection and separation technologies
Sanger sequencing is still very labor intensive
©2001 Timothy G. Standish
 Labeling and Detection
The most significant advance in labeling
has been the production of
electrophoretically neutral dyes that
fluoresce at specific wavelengths when
excited by laser-produced light over a
very narrow range of wavelengths
These dyes, when attached to primers
allow detection down to 15 attomoles
(10-18)
That’s less than 107 molecules! ©2001 Timothy G. Standish
 Applied Biosystems
Applied Biosystems (ABI) has developed fluorescent dye
systems further and improved methods for loading and
electrophoresis
Four dyes each of which fluoresce at a different
wavelength, but having about the same impact on
electrophoritic mobility can be used to label either primers
or the nucleotides that terminate a reaction
If terminator dyes are used, the entire sequencing reaction
is reduced to one tube from 4 in conventional Sanger
sequencing
Instead of polyacrylamide slab gels, a single capillary can
be used with a liquid polymer that is replaced after each
individual run ©2001 Timothy G. Standish
Replication Using Dye Terminators
3’AATAGCATAACGTTAACGTTACGCTAT5’
5’TTATCG
5’TTATCGTACCATAATTGCA
5’TTATCGTACCATAATT
5’TTATCGTACCAC
5’TTATCGTA
5’TTATCGTA
5’TTATCGTAT As the base at the
5’TTATCGTATT end of each
5’TTATCGTATTG fragment is clearly
5’TTATCGTATTGC marked with a
5’TTATCGTATTGCA
5’TTATCGTATTGCAA unique fluorescent
5’TTATCGTATTGCAAT dye, the entire
5’TTATCGTATTGCAATT reaction can be
5’TTATCGTATTGCAATTG done in a single
5’TTATCGTATTGCAATTGC tube
5’TTATCGTATTGCAATTGCA
©2001 Timothy G. Standish
 ABI Prism 310 System
ATTGC A

Capillary
Liquid
polymer
…..

Laser
Heat plate
-
Beam
splitter Sequencing
Window reaction
Detectors
+ ©2001 Timothy G. Standish
 The State of the Art
The ABI Prism 310 (1 capillary), 3100 (16
capillaries) and 3700 (96 capillaries) represent the
current state of the art in automated sequencing
machines
A single ABI Prism 377 slab gel sequencer can run
115,000 bases per day!
The 3100 can run up to 184,000 bases per day
The 3700 can run up to 1,104,000 bases per day
Large sequencing facilities, like Celera, have
factories full of these machines which can run 24
hours a day with very little down time for routine
maintenance
©2001 Timothy G. Standish