Gene Cloning

Sequencing DNA 181
double-stranded copy. The double-stranded genome will then direct the
expression of viral genes and act as template for the synthesis of singlestranded
viral genome that is packaged into the virion prior to release of the
virion from the bacteria. Because M13 is a helical virus its genome size is not
restricted. So in a culture of E. coli infected by bacteriophage M13, the bacterial
cells will contain double-stranded viral genome, whereas the culture
medium will contain the virion with its single-stranded DNA genome. It is
therefore relatively easy to isolate both single and double-stranded forms of
the viral genome by separating the bacterial cells from the culture medium
by centrifugation. All these properties of M13 make it ideal for producing
single-stranded DNA. Plasmid vectors were developed that contain M13
sequences, and these vectors can be purified from host E. coli cells in the
double-stranded form and are therefore tractable to the standard cloning
procedures discussed in Chapter 3. They will, however, also direct the synthesis
of a single-stranded copy of the plasmid sequence which is packaged
into a viral particle and excreted into the culture medium. Single-stranded
DNA can therefore be purified from the growth medium.
Cloning into M13-derived vectors is a very robust procedure for obtaining
single-stranded DNA for sequencing. However, assuming that your
DNA to be sequenced is already present in a standard plasmid vector, it
requires a sub-cloning step to transfer your sequence into the M13-based
vector. It was therefore desirable to develop a procedure that would allow
the direct sequencing of double-stranded DNA by generating singlestranded
template in the reaction by either chemical or heat treatment.
7.5 Modifications of the Original Sanger Protocol
As it is based on de novo synthesis of DNA, the Sanger chain termination
method of DNA sequencing requires a DNA polymerase; initial protocols
used the Klenow fragment (Box 3.5). As sequencing protocols developed
the Klenow fragment has been replaced with other DNA polymerases (Box
3.5). T7 DNA polymerase first replaced the Klenow fragment in sequencing
protocols because it facilitated the development of more robust protocols
in which double-stranded DNA templates could be sequenced. These protocols
generate a single-stranded template by alkali denaturation of the
template DNA. The primer is added to the denatured template at neutral
pH and the sequencing protocol completed as discussed previously and as
shown in Figures 7.2 and 7.3.
The development of thermostable DNA polymerase has allowed the use
of heat denaturation to generate single-stranded template (as in the polymerase
chain reaction). The use of Taq DNA polymerase has made possible
the use of PCR-based sequencing protocols. Another advantage of the
polymerase chain reaction is that it can be exploited to generate sufficient
template for sequencing without the need to clone a DNA fragment into a
plasmid vector. This does facilitate the direct sequencing of genomic DNA,