DNA SEQUENCING

DAN BLAST
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DNA
• Stands for
Deoxyribonucleic acid
• Made up of subunits called
nucleotides
• Nucleotide made of:
1. Phosphate group
2. 5-carbon sugar
3. Nitrogenous base

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Phosphate
DNA NUCLEOTIDE
Group

O
5
O=P-O CH2
O
O
N
Nitrogenous base
C4 C1 (A, G, C, or T)

Sugar
(deoxyribose)
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 PENTOSE SUGAR
4

• Carbons are numbered clockwise 1’ to 5’

5
CH2

C4 C1
Sugar
(deoxyribose)

C3 C2
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 DNA 5

5 O 3

3 O
P 5 P
5 O
1 G C 3
2
4 4
2 1
3 5
P O
P
5
T A 3
O

O
5
P 3 P
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 ANTIPARALLEL 6

STRANDS
• One strand of
DNA goes from
5’ to 3’ (sugars)
• The other strand
is opposite in
direction going 3’
to 5’ (sugars)

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NITROGENOUS BASES
• Double ring PURINES
Adenine (A)
Guanine (G) A or G

• Single ring PYRIMIDINES

Thymine (T)
Cytosine (C) T or C

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 BASE-PAIRINGS 8

• Purines only pair with

Pyrimidines
• Three hydrogen bonds
required to bond Guanine &
Cytosine
3 H-bonds

G C

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 9

•TWO HYDROGEN BONDS

ARE REQUIRED TO BOND
ADENINE & THYMINE

T A

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QUESTION:
•If there is 30% Adenine, how much
Cytosine is present?

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 ANSWER:
11

• There would be 20%

Cytosine
• Adenine (30%) = Thymine
(30%)
• Guanine (20%) = Cytosine
(20%)
• Therefore, 60% A-T and
40% C-G
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 DETERMINING DNA SEQUENCE
• Originally 2 methods were invented around 1976, but
only one is widely used: the chain-termination method
invented by Fred Sanger.
• The other method is Maxam-Gilbert chemical degradation
method, which is still used for specialized purposes, such as
analyzing DNA-protein interactions.
• More recently, several cheaper and faster alternatives
have been invented. It is hard to know which of these
methods, or possibly another method, will ultimately
become standard. We will discus two of them: 454
pyrosequencing and Illumina/Solexa sequencing
 SANGER SEQUENCING
• Uses DNA polymerase to synthesize a second
DNA strand that is labeled. DNA polymerase
always adds new bases to the 3’ end of a primer
that is base-paired to the template DNA.
• DNA polymerase is modified to eliminate its
editing function
• Also uses chain terminator nucleotides: dideoxy
nucleotides (ddNTPs), which lack the -OH group
on the 3' carbon of the deoxyribose. When DNA
polymerase inserts one of these ddNTPs into the
growing DNA chain, the chain terminates, as
nothing can be added to its 3' end.
 SEQUENCING REACTION
• The template DNA is usually single stranded
DNA, which can be produced from plasmid
cloning vectors that contain the origin of
replication from a single stranded
bacteriophage such as M13 or fd. The
primer is complementary to the region in the
vector adjacent to the multiple cloning site.

• Sequencing is done by having 4 separate

reactions, one for each DNA base.
• All 4 reactions contain the 4 normal dNTPs,
but each reaction also contains one of the
ddNTPs.
• In each reaction, DNA polymerase starts
creating the second strand beginning at the
primer.
• When DNA polymerase reaches a base for
which some ddNTP is present, the chain will
either:
• terminate if a ddNTP is added, or:
• continue if the corresponding dNTP is
added.
• which one happens is random, based
on ratio of dNTP to ddNTP in the tube.
• However, all the second strands in, say, the
A tube will end at some A base: you get a
collection of DNAs that end at each of the
A's in the region being sequenced.
ELECTROPHORESIS
• The newly synthesized DNA from
the 4 reactions is then run (in
separate lanes) on an
electrophoresis gel.
• The DNA bands fall into a
ladder-like sequence, spaced
one base apart. The actual
sequence can be read from the
bottom of the gel up.
• Automated sequencers use 4
different fluorescent dyes as
tags attached to the dideoxy
nucleotides and run all 4
reactions in the same lane of
the gel.
• Today’s sequencers use
capillary electrophoresis instead
of slab gels.
• Radioactive nucleotides (32P)
are used for non-automated
sequencing.
• Sequencing reactions usually
produce about 500-1000 bp of
good sequence.