Biomedical Engineering Reference
In-Depth Information
the same aberrations and the greatest problem with
them arises when the DNA being cloned contains
tandem arrays of repeated sequences. The problem
is particularly acute when the tandem array is sev-
eral times larger than the allowable size of a cosmid
insert. Potential advantages of the BAC and PAC
systems over YACs include lower levels of chimerism
(Hartl
et al.
1994, Sternberg 1994), ease of library
generation and ease of manipulation and isolation of
insert DNA. BAC clones seem to represent human
DNA far more faithfully than their YAC or cosmid
counterparts and appear to be excellent substrates
for shotgun sequence analysis, resulting in accurate
contiguous sequence data (Venter
et al.
1996).
resistance gene inserted into the M13
ori
region and
carries a mutation in the
gII
gene (Vieira & Messing
1987). M13KO7 can replicate on its own. However,
in the presence of a phagemid bearing a wild-type
origin of replication, single-stranded phagemid is
packaged preferentially and secreted into the cul-
ture medium. DNA purified from the phagemids can
be used directly for sequencing.
Expression vectors
Expression vectors are required if one wants to pre-
pare RNA probes from the cloned gene or to purify
large amounts of the gene product. In either case,
transcription of the cloned gene is required. Although
it is possible to have the cloned gene under the con-
trol of its own promoter, it is more usual to utilize a
promoter specific to the vector. Such vector-carried
promoters have been optimized for binding of the
E.
coli
RNA polymerase and many of them can be regu-
lated easily by changes in the growth conditions of
the host cell.
E. coli
RNA polymerase is a multi-subunit enzyme.
The core enzyme consists of two identical
Specialist-purpose vectors
Vectors that can be used to make
single-stranded DNA for sequencing
Whenever a new gene is cloned or a novel genetic
construct is made, it is usual practice to sequence all
or part of the chimeric molecule. As will be seen later
(p. 120), the Sanger method of sequencing requires
single-stranded DNA as the starting material.
Originally, single-stranded DNA was obtained by
cloning the sequence of interest in an M13 vector
(see p. 60). Today, it is more usual to clone the
sequence into a pUC-based phagemid vector which
contains the M13
ori
region as well as the pUC
(Col E1) origin of replication. Such vectors norm-
ally replicate inside the cell as double-stranded
molecules. Single-stranded DNA for sequencing can
be produced by infecting cultures with a helper
phage such as M13K07. This helper phage has the
origin of replication of P15A and a kanamycin-
α
subunits
and one each of the
subunits. The core
enzyme is not active unless an additional subunit,
the
β
and
β′
factor, is present. RNA polymerase recognizes
different types of promoters depending on which
type of
σ
factor is attached. The most common pro-
moters are those recognized by the RNA polymerase
with
σ
70
promoters from
E. coli
have been analysed and a compilation of over 300 of
them can be found in Lisser and Margalit (1993). A
comparison of these promoters has led to the formu-
lation of a consensus sequence (Fig. 5.6). If the tran-
scription start point is assigned the position
70
. A large number of
σ
σ
+
1 then
-35 Region
-10 Region
123456789 1011121314151617
CONSENSUS
lac
trp
λ
P
L
rec A
tac
I
tac
II
•••
T
T
T
T
T
T
T
T
T
T
T
T
T
T
G
T
G
G
G
G
G
A
A
A
A
A
A
A
C
C
C
C
T
C
C
A
A
A
A
A
A
A
••••••••• ••••••••
T
T
T
G
T
T
T
A
A
T
A
A
A
T
T
T
A
T
T
T
T
A
A
A
A
A
A
A
A
T
C
C
A
A
A
T
T
T
T
T
T
T
••
G
C
G
C
C
C
G
T
T
A
T
T
C
G
G
C
G
G
C
A
T
C
A
A
T
T
A
T
T
T
T
T
A
G
T
T
T
A
A
T
A
A
A
A
T
A
A
A
T
T
A
T
T
T
G
C
C
G
C
C
C
A
C
A
A
A
T
T
A
A
T
T
TC
C
C
G
C
G
T
C
C
G
G
A
G
A
G
A
G
A
G
T
G
A
C
C
C
A
C
C
T
T
G
C
T
T
C
A
G
A
C
A
G
G
T
G
G
G
G
A
G
T
G
G
T
G
A
G
T
T
Fig. 5.6
The base sequence of the
−10 and −35 regions of four natural
promoters, two hybrid promoters and
the consensus promoter.
C
