DNA sequencing: methods

Why sequence DNA?

• All genes available for an organism to use

-- a very important tool for biologists
• Not just sequence of genes, but also
positioning of genes and sequences of
regulatory regions

• New recombinant DNA constructs must be

sequenced to verify construction or
positions of mutations
• Etc.
History of DNA sequencing
History of DNA sequencing

MC chapter 12
 Methods of sequencing

A. Sanger dideoxy (primer extension/chain-

termination) method: most popular protocol for
sequencing, very adaptable, scalable to large
sequencing projects

B. Maxam-Gilbert chemical cleavage method: DNA is

labelled and then chemically cleaved in a
sequence-dependent manner. This method is not
easily scaled and is rather tedious

C. Pyrosequencing: measuring chain extension by

pyrophosphate monitoring
for dideoxy sequencing you
need:

1) Single stranded DNA template

2) A primer for DNA synthesis
3) DNA polymerase
4) Deoxynucleoside triphosphates and
dideoxynucleotide triphosphates
 Primers for DNA sequencing

• Oligonucleotide primers can be synthesized by

phosphoramidite chemistry--usually designed
manually and then purchased

• Sequence of the oligo must be complimentary

to DNA flanking sequenced region

• Oligos are usually 15-30 nucleotides in length

 DNA templates for sequencing:

• Single stranded DNA isolated from

recombinant M13 bacteriophage
containing DNA of interest
• Double-stranded DNA that has been
denatured
• Non-denatured double stranded DNA
(cycle sequencing)
One way for obtaining single-stranded DNA from a
double stranded source--magnets
 Reagents for sequencing:
DNA polymerases

• Should be highly processive, and

incorporate ddNTPs efficiently

• Should lack exonuclease activity

• Thermostability required for “cycle

sequencing”
Sanger dideoxy sequencing--basic
method

3’ Single stranded DNA 5’

5’ 3’

a) Anneal the primer

 Sanger dideoxy sequencing: basic
method
5’

Direction
b) Extend the of DNA
primer with DNA polymeras
polymerase in e travel
the presence of
3’
all four dNTPs,
with a limited
amount of a
dideoxy NTP
(ddNTP)
DNA polymerase incorporates ddNTP in a
template-dependent manner, but it works best
if the DNA pol lacks 3’ to 5’ exonuclease
(proofreading) activity
 Sanger dideoxy sequencing: basic
method

3’ T TT T 5’

5’ 3’
ddATP in the
ddA
reaction:
ddA anywhere there’s
a T in the
ddA
template strand,
ddA occasionally a ddA
will be added to
the growing strand
 How to visualize DNA fragments?

• Radioactivity
– Radiolabeled primers (kinase with 32P)
– Radiolabelled dNTPs (gamma 35S or 32P)

• Fluorescence
– ddNTPs chemically synthesized to contain
fluors
– Each ddNTP fluoresces at a different
wavelength allowing identification
 Analysis of sequencing products:

Polyacrylamide gel electrophoresis--good

resolution of fragments differing by a
single dNTP
 DNA sequencing gels: old school
Different ddNTP used in
separate reactions
Analyze
sequencing
products by gel
electrophoresis,
autoradiography

Radioactively labelled primer or

dNTP in sequencing reaction
 cycle sequencing: denaturation
occurs during temperature cycles
94°C:DNA denatures

45°C: primer anneals

60-72°C: thermostable
DNA pol extends primer

Repeat 25-35 times

Advantages: don’t need

a lot of template DNA

Disadvantages: DNA pol

may incorporate ddNTPs
poorly
Animation of cycle sequencing: see
http://www.dnai.org/

Click on:
“manipulation”
“techniques”
“sorting and sequencing”
An automated sequencer

The output
 Current trends in sequencing:
It is rare for labs to do their own sequencing:
--costly, perishable reagents
--time consuming
--success rate varies

Instead most labs send out for sequencing:

--You prepare the DNA (usually plasmid, M13, or PCR

product), supply the primer, company or university
sequencing center does the rest

--The sequence is recorded by an automated sequencer

as an “electropherogram”
 BREAK UP THE GENOME,
PUT IT BACK TOGETHER

Assemble sequences
~160 kbp by matching overlaps

BAC sequence
~1 kbp

BAC overlaps give genome sequence

 Sequencing large pieces of DNA:
the “shotgun” method
• Break DNA into small pieces (typically sizes of
around 1000 base pairs is preferable)
• Clone pieces of DNA into M13
• Sequence enough M13 clones to ensure complete
coverage (eg. sequencing a 3 million base pair
genome would require 5x to 10x 3 million base
pairs to have a reliable representation of the
genome)
• Assemble genome through overlap analysis using
computer algorithms, also “polish” sequences
using mapping information from individual clones,
characterized genes, and genetic markers
• This process is assisted by robotics
 Sequencing done by TIGR (Maryland) and
The Sanger Institute (Cambridge, UK)

“Here we report an analysis of the genome sequence

of P. falciparum clone 3D7, including descriptions of
chromosome structure, gene content, functional
classification of proteins, metabolism and transport,
and other features of parasite biology.”
Sequencing strategy
A whole chromosome shotgun sequencing
strategy was used to determine the genome
sequence of P. falciparum clone 3D7. This
approach was taken because a whole genome
shotgun strategy was not feasible or cost-
effective with the technology that was
available at the beginning of the project. Also,
high-quality large insert libraries of (A - T)-rich
P. falciparum DNA have never been
constructed in Escherichia coli, which ruled out
a clone-by-clone sequencing strategy. The
chromosomes were separated on pulsed field
gels, and chromosomal DNA was extracted…
 The shotgun sequences were assembled into
contiguous DNA sequences (contigs), in some
cases with low coverage shotgun sequences of
yeast artificial chromosome (YAC) clones to assist
in the ordering of contigs for closure. Sequence
tagged sites (STSs)10, microsatellite markers11,12
and HAPPY mapping7 were also used to place and
orient contigs during the gap closure process. The
high (A /T) content of the genome made gap
closure extremely difficult7–9.

Chromosomes 1–5, 9 and 12 were closed,

whereas chromosomes 6–8, 10, 11, 13 and 14
contained 3–37 gaps (most less than 2.5 kb) per
chromosome at the beginning of genome
annotation. Efforts to close the remaining gaps are
continuing.
Methods: Sequencing, gap closure and annotation
The techniques used at each of the three
participating centres for sequencing, closure and
annotation are described in the accompanying Letters7–
9
. To ensure that each centres’ annotation procedures
produced roughly equivalent results, the Wellcome
Trust Sanger Institute (‘Sanger’) and the Institute for
Genomic Research (‘TIGR’) annotated the same100-kb
segment of chromosome 14. The number of genes
predicted in this sequence by the two centres was 22
and 23; the discrepancy being due to the merging of
two single genes by one centre. Of the 74 exons
predicted by the two centres, 50 (68%) were identical, 9
(2%) overlapped, 6 (8%) overlapped and shared one
boundary, and the remainder were predicted by one
centre but not the other. Thus 88% of the exons
predicted by the two centres in the 100-kb fragment
were identical or overlapped.
Previous sequencing techniques: one DNA molecule at a
time
Needed: many DNA molecules at a time -- arrays

One of these: “pyrosequencing”

Cut a genome to DNA fragments 300 - 500 bases long

Immobilize single strands on a very small plastic bead

(one piece of DNA per bead)

Amplify the DNA on each bead to cover each bead to

boost the signal

Separate each bead on a plate with up to 1.6 million

wells
Sequence by DNA polymerase -dependent chain
extension, one base at a time in the presence of a
reporter (luciferase)

Luciferase is an enzyme that will emit a photon of light

in response to the pyrophosphate (PPi) released upon
nucleotide addition by DNA polymerase

Flashes of light and their intensity are recorded