Sequencing and mutagenesis - Principles of Gene Manipulation - page 122

Sequencing gel autoradiograph

A C G T

Electr oph oresis

T
C
G
C
A
G
T
C
C
T
A
G
C
T
T
A
G
C
G
G '

3'

Fig. 7.3 Enlarged autoradiograph of a
sequencing gel obtained with the chain-
terminator DNA sequencing method.

Sal I

Eco R I Bam I Sal I Pst I Bam I Eco R I

5 ′ 3 ′

ATGACCATGATTACGAATTCCCCGGATCCGTCGACCTGCAGGTCGACGGATCCGGGGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGG
TTGCAGCACTGACCC

3 ′ 5 ′

Acc I Hin c II Acc I Hin c II

Primer
oligonucleotide

Synthesis of
DNA in
sequencing
reaction

1 2 3 4 Direction of

lac translation

Start of
translation of
lac α fragment

Fig. 7.4 Sequence of M13 mp7 DNA in the vicinity of the multipurpose cloning region. The upper sequence is that of M13 mp7
from the ATG start codon of the β-galactosidase α fragment, through the multipurpose cloning region and back into the β-
galactosidase gene. The short sequence at the right-hand side is that of the primer used to initiate DNA synthesis across the
cloned insert. The numbered boxes correspond to the amino acids of the β-galactosidase fragment.

dideoxynucleotides as substrates (Fig. 7.1). Once the
analogue is incorporated at the growing point of the
DNA chain, the 3

the Klenow fragment of DNA polymerase is used
because this lacks the 5 exonuclease activity

associated with the intact enzyme. Initiation of DNA
synthesis requires a primer and this is usually a
chemically synthesized oligonucleotide which is
annealed close to the sequence being analysed.

′→ 3 ′

end lacks a hydroxyl group and
is no longer a substrate for chain elongation. Thus,
the growing DNA chain is terminated, i.e. dideoxy-
nucleotides act as chain terminators. In practice,

′