Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 11

SEQUENCING THE FIFTH BASE

391

Figure 11.22

Genomic DNA sequencing can be used to find the location of methylated bases.

(a )

Basic scheme for

PCR-mediated genomic Maxam-Gilbert DNA sequencing. (

b ) A methylated base

prevents normal

Maxam-Gilbert cleavage and thus alters the appearance of the DNA sequencing

ladder. Adapted from Riggs et al. (1991).

nomic DNA is fragmented chemically as in the Maxam-Gilbert procedure. A sequence-

specific primer is annealed to the DNA after this

cleavage and extended up to the site

of

the cleavage. This creates a ligatable end. A splint is ligated onto all the genomic DNA

pieces. Then primers are used to extend the DNA

past the unique region of the splint,

as

we described earlier for single-sided PCR reactions in Chapter 4. Finally two primers are

used for exponential amplification. The sizes of the amplified products reveal where the

original cleavages were in the DNA. If all bases in the genome are accessible to the spe-

cific cleavage reactions used, then a perfectly normal sequencing ladder should result.

To find the location of

m C in genomic sequence, one takes advantage

of the fact that

this base renders DNA resistant to cleavage by the normal C-specific reaction used in the

Maxam-Gilbert method. The result, is that the locations of the methylated C’s drop out of

the sequencing ladder (Fig. 11.22

b ). If the target sequence is already completely known

except for the sites of methylation, this is all the information one needs. If not, one can al-

ways repeat the sequencing on a cloned

sample to

confirm the location of the

m C’s by

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