Some Additional Tools for the Molecular Biologist - Insect Molecular Genetics - page 224

Figure 6.8 Outline of the infection cycle of the bacteriophage M13. The single-stranded phage
attaches to the F pilus of E. coli , injects its DNA into the host, and begins to produce ≈ 100 copies of
double-stranded (RF) molecules. DNA replication then shifts to producing ss DNA molecules that
are extruded through the host cell wall, during which time they are coated with a capsid protein
coat. M13 has been engineered as a vector and is used to produce ss DNA molecules, a technology
that is particularly useful in DNA sequencing reactions.
until ≈ 100 RF molecules are produced within the cell ( Figure 6.8 ). The replica-
tion of the RF then becomes asymmetric due to the accumulation of a viral-
encoded binding protein that is specific to single-stranded (ss) DNA. The binding
protein binds to the M13 DNA and prevents synthesis of a complementary
strand. Subsequently, only ss DNA is synthesized and extruded from the host
cell. As the ss M13 DNA molecules move through the E. coli cell membrane,
the DNA-binding protein is removed and the M13 DNA is coated with capsid
protein.
M13 has many advantages as a vector. First, ss DNA is required in several
applications, including the dideoxy DNA sequencing method (described in
Chapter 7). Second, ss M13 vectors allow the genetic engineer to combine
cloning, amplification, and strand separation of a ds DNA fragment in one
operation. Third, because the phage DNA is replicated in a ds circular (RF) inter-
mediate stage, the RF DNA can be purified and manipulated just like a plasmid.
Fourth, both RF and ss DNA will transfect competent E. coli cells and yield either
plaques or infected colonies. Fifth, it is possible to package DNA up to 6 times
the length of the wild-type M13 DNA.