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the chain is terminated at each site where Ts occur. These DNA molecule popula-
tions can be visualized by gel electrophoresis.
The base sequence is visualized by running the radiolabeled DNA fragments
from the four reactions on an acrylamide gel in four adjacent lanes. Each reac-
tion tube will produce a series of bands, with each band representing a popula-
tion of molecules at which the DNA molecule was terminated by incorporating
a ddNTP. The banding pattern in the four lanes was visualized on an X-ray film
( Figure 7.4 ). The two strands should be sequenced independently to reduce
errors generated by artifacts in the sequence reactions or inadequate resolution
of regions of the sequence on the gel.
There are several different protocols for sequencing DNA by the dideoxy
chain-termination method ( Ambrose and Pless 1987, Barnes 1987, Mierendorf
and Pfeffer 1987, Tabor and Richardson 1987, Howe and Ward 1989, Sambrook
et al. 1989 ). Variables include such factors as whether the DNA is double- or sin-
gle-stranded and whether the DNA is sequenced directly from double-stranded
plasmid DNA or after subcloning into a single-stranded phage such as M13.
Today, the ddNTPs are labeled by one of four different fluorescent dyes. The
results are shown as a chromatogram with a series of peaks of A, T, C, and G
labeled with different colors.
Both Sanger and Maxam and Gilbert sequencing methods generate sets
of DNA molecules that share a common end that is determined by the prim-
ers but that vary in their length at the other end. Both methods also originally
used radioactive labeling to visualize the results. Once the DNA was electropho-
resed, the gel was dried onto paper and exposed to X-ray film. The autoradio-
gram produced displayed a ladder of bands representing the migration position
of the different radiolabeled DNA segments. For example, the sequence of 20
nucleotides can be read to the right of the four lanes in Figure 7.4 .
Both the Sanger and Maxam and Gilbert sequencing methods originally used
very thin polyacrylamide gels to discriminate between nucleic-acid molecules
that may differ in length by only one nucleotide. A sequencing gel is a high-
resolution gel containing 6-20% polyacrylamide and 7M urea. The DNA to be
analyzed is denatured before electrophoresis by heating it to 80°C in a buffer
containing formamide, which lowers the melting temperature of ds DNA mol-
ecules. The urea minimizes DNA secondary structure, which could affect mobil-
ity of the DNA through the gel. The gel is run using sufficient power so that
it is heated to about 50°C, which also minimizes DNA secondary structure.
The length of the DNA molecule determines its rate of migration. The shorter
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