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fragments migrate more quickly than the longer, thus ending up nearer the bot-
tom of the gel ( Davies 1982, Sealey and Southern 1982, Howe and Ward 1989 ).
If the template DNA is a subclone that was inserted into the multiple-cloning
site of a vector, then the primer almost always is an oligonucleotide complemen-
tary to sequences flanking the multiple-cloning site. This allows any fragment
cloned into the multiple-cloning sites to be sequenced using the same primer.
Most vectors have the lacZ sequences flanking the multiple cloning sites. Thus,
an oligonucleotide primer directed to this sequence, which is 16 or 17 nucleo-
tides long, is commonly used and called the M13 universal primer . This primer
is designed to be complementary to the strand of DNA packaged into M13 or
into any plasmid vector containing an M13 origin of replication. Methods also
have been developed for direct sequencing from denatured plasmid DNA
( Mierendorf and Pfeffer 1987 ), eliminating the need to isolate or subclone DNA
fragments.
7.4 The Maxam and Gilbert Sequencing Method
Maxam and Gilbert DNA sequencing is called the chemical-cleavage method
( Maxam and Gilbert 1977, 1980 ). It uses chemical reagents to generate base-spe-
cific cleavages of the DNA to be sequenced ( Figure 7.5 ). It was less-often used,
in part because the chemicals are toxic and the methods are labor-intensive. The
primary advantage of this method is that DNA sequences are obtained from the
original DNA molecule and not from a synthesized copy. Thus, one can analyze
DNA modifications such as methylation and study DNA secondary structure and
the interaction of proteins with DNA.
To start, one needs pure DNA that has been cut by restriction endonucle-
ases to generate DNA of specific length and with known sequences at one
end. Each DNA fragment then can be radioactively labeled at one end with a
32 P-phosphate group in sufficient quantity that at least four different chemi-
cal reactions can be carried out. Next, specific bases in the DNA fragment
are altered in four separate chemical reactions. For example, guanine (G) is
methylated by dimethylsulfate. Each reaction is carried out in a manner that
limits the reaction so that, for example, only one G is modified per several
hundred Gs. The altered G is then removed, in a subsequent step, by cleavage at
the modification points with piperdine. The result is a set of end-labeled frag-
ments of different lengths that will show up as a ladder of bands on the gel
because the reaction was limited and not all the Gs were altered in the reaction.
Four different reaction samples (one for A + G, G, T + C, and C) are then run
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