Ten micrograms of a decanucleotide-pair Hpal restriction fragment were
isolated from the double-stranded DNA chromosome of a small virus.
Octanucleotide poly(A) tails were then added to the \(3^{\prime}\) ends of both
strands using terminal transferase and dATP; that is,
\\[\begin{array}{l}
5^{\prime}-\mathrm{X} \times \mathrm{X} \times \mathrm{X} \times \mathrm{X}
\times \mathrm{X} \times-3^{\prime} \\
3^{\prime}-\mathrm{X}^{\prime} \mathrm{X}^{\prime} \mathrm{X}^{\prime}
\mathrm{X}^{\prime} \mathrm{X}^{\prime} \mathrm{X}^{\prime}
\mathrm{X}^{\prime} \mathrm{X}^{\prime} \mathrm{X}^{\prime}
\mathrm{X}^{\prime}-5^{\prime}\end{array}\\]
\(\downarrow\) terminal transferase, dATP
\\[5-\mathrm{XXXXXXXXXXAAAAAAAA-3^{\prime }}\\]
\(3^{\prime}\) -AAAAAAAAX'X'X'X'X'X'X'X'X'X'-5'
where \(X\) and \(X^{\prime}\) can be any of the four standard nucleotides, but
\(X^{\prime}\) is always complementary to \(X\)
The two complementary strands ("Watson" strand and "Crick" strand) were then
separated and sequenced by the \(2^{\prime}, 3^{\prime}\) -dideoxyribonucleoside
triphosphate chain termination method. The reactions were primed using a
synthetic poly(T) octamer; that is,
Watson strand
\(3^{\prime}-\mathrm{A} \mathrm{A} \mathrm{A} \mathrm{A} \mathrm{A} \mathrm{A}
\mathrm{A} \mathrm{A} \mathrm{X}^{\prime} \mathrm{X}^{\prime}
\mathrm{X}^{\prime} \mathrm{X}^{\prime} \mathrm{X}^{\prime}
\mathrm{X}^{\prime} \mathrm{X}^{\prime} \mathrm{X}^{\prime}
\mathrm{X}^{\prime} \mathrm{X}^{\prime}-5^{\prime}\)
\(5^{\prime}-\mathrm{T} \mathrm{T} \mathrm{T}\) T \(\mathrm{T}\) T \(\mathrm{T}\)
T-OH
Crick strand
\\[5^{\prime}-X X X X X X X X X X A A A A A A A A-3^{\prime}\\]
HO-T T T T T T-5'
Two DNA sequencing reactions were carried out. Reaction 1 contained the Watson
strand template/primer shown above; reaction 2 contained the Crick strand
template/primer. Both sequencing reactions contained DNA polymerase and all
other substrates and components required for DNA synthesis in vitro plus the
standard four \(2^{\prime}, 3^{\prime}\) -dideoxyribonucleoside triphosphate
chain terminators ddGTP, ddCTP, ddATP, and ddTTY each labeled with a different
fluorescent dye. The dyes fluoresce at different wavelengths, which are
recorded by a photocell as the products of the reactions are separated by
capillary gel electrophoresis (see Figure 14.17 ). In the standard sequencing
reaction, the chains terminating with ddG fluoresce dark blue (peaks appear
black in the computer printouts), those terminating with ddC fluoresce light
blue, those terminating with ddA fluoresce green, and those terminating with
ddT fluoresce red. The computer printout for sequencing reaction \(1,\) which
contained the Watson strand as template, is as follows.
Draw the predicted computer printout for reaction 2 which contained the Crick
strand as template, in the following box. Remember that all DNA synthesis
occurs in the \(5^{\prime} \rightarrow 3^{\prime}\) direction and that the
sequence of the nascent strand reads \(5^{\prime}\) to \(3^{\prime}\) from left to
right in the printout.
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