self-ligate without insert. Furthermore, not all bacteria used in the transformation
step will have received a vector molecule. It can therefore be that there is only a small
proportion of cells that contain a vector with insert in the growing cell population.
There are different strategies to cope with this problem, but normally one tries to
make use of genes in the original vector that can be used as selection markers. Fig-
ure 4.3 shows pBR322, a typical cloning vector. Apart from a DNA sequence that en-
ables the cell machinery to replicate the plasmid (ori), it also contains two genes for
resistance against the antibiotics ampicillin and tetracycline. If the DNA fragment is
cloned into a restriction site that lies within one of the resistance genes, e.g., the
Bam HI site, simple selection steps can be used to end up with cells that contain the
desired construct. For this purpose the bacteria are grown in a medium that contains
ampicillin so that only cells that carry the plasmid can survive. The next step is more
complicated since we are interested in all those bacteria that contain a vector with a
nonfunctional tetracycline gene (caused by an insert). The cells are plated in high di-
lution on the surface of an agar plate, where each individual cell forms a colony. After
the colonies become visible, some bacteria of each colony are copied onto a second
agar plate by a stamping technique (which preserves the spatial arrangement of colo-
nies). This second plate contains tetracycline; therefore, only those cells with an in-
tact resistance gene can grow. By comparing the colony pattern of the two plates, it is
now possible to identify those colonies that exist on the first plate but not on the sec-
ond. These are the colonies that we are interested in.
For many years plasmid vectors have been used very successfully. However, there
is an upper size limit for the DNA one can clone into such a vector. Above 10 kb, the
cloning efficiency declines so much that other vectors are required. The following ta-
ble lists some of the types of vectors currently used. Lambda is a linear bacteriophage
of approximately 48 kb, and up to 20 kb of the original phage DNA can be replaced
by foreign DNA. Cosmids are artificial constructs that combine some features of the
phage lambda and of the classical plasmids. The advantage is that fragments up to
45 kb can be cloned. For really large fragments of up to one million base pairs, yeast
artificial chromosomes (YACs) have been developed (Burke et al. 1987), which are
now gradually being replaced by bacterial artificial chromosomes (BACs) (Shizuya
et al. 1992). BACs are based on the naturally occurring F-plasmid, which itself is
around 100 kb in length. While the copy number per cell of most smaller plasmids
is rather large, the F-plasmid and the derived BACs are maintained at only one to
two copies per cell. This reduces the risk of unwanted recombination events between
different copies and contributes to the stability of such large inserts.
115
4.1 Elementary Techniques
3 Fig. 4.3 pBR322 is a circular plasmid of 4.3 kb
that is often used as cloning vector. The diagram
on the top shows several important genetic ele-
ments of the plasmid. ORI marks a region of
DNA that controls the replication of the plas-
mid; it is the origin of replication. The boxes
represent genes that confer resistance to the
plasmid for the antibiotics ampicillin (APr) and
tetracycline (TCr). P-A and P-R are the promoters
of the resistance genes, and the lines and text
mark recognition sites for the corresponding
restriction enzyme. The lower part shows
pBR322 after a restriction fragment has been
inserted into the Bam HI restriction site.