Gene Cloning

90 Gene Cloning
which is 6.3 Mb, to have a 99% chance of finding any particular gene, you
would need to have 7251 clones. Notice that because the calculation is
based on probabilities and you have no way of ensuring that each clone in
the library is unique, you need considerably more clones than the 1500
restriction fragments BamHI would cut the Pseudomonas genome into.
With 10,876 clones the probability goes up to 99.9% and it would probably
not be worth increasing the library size any further than this.
Q4.4. How many clones would you need in a genomic library made from
(a) E. coli or (b) Saccharomyces cerevisiae to have a 99% chance of cloning a
particular gene? Assume an average fragment size of 4000 kb as before.
Q4.5. How many clones of E. coli would you need to have a 99.9% chance
of cloning a particular gene?
4.5 Some DNA Fragments are Under-represented in
Genomic Libraries
Some stretches of the genome are harder to clone than others because of
the burden placed on the host cell by the genes encoded on them. A good
example of this is genes for proteins which are located in membranes. If
such proteins are over-expressed they can often cause damage to the
membranes of the host bacterium, and reduce its viability. Cloned genes
may be expressed at higher levels than in their normal setting because they
have been cloned into a multiple copy number plasmid and also possibly
removed from the normal regulation mechanisms. This may also place an
unacceptable burden on the host bacterium.
Larger DNA fragments are also harder to clone than small ones, mostly
because as they are big there is a greater chance that they will contain
sequences which make them unstable or which place a burden on the host
cell. Because restriction enzyme recognition sites occur randomly throughout
the genome a particular gene may be located on a very large fragment,
which may be under-represented in the genomic library. Some stretches of
DNA are intrinsically less stable when cloned than others, often because they
contain repetitive sequences which may cause mistakes during DNA replication.
These regions are also likely to be under-represented in DNA libraries.
The probability calculations are based on the assumption that any particular
piece of the genome is just as likely as any other piece to be successfully
cloned. This is clearly not true; in practice most workers will use a
library with at least two or three times as many clones as are theoretically
needed, to give a high probability of finding a particular gene.
4.6 Using Partial Digests to Make a Genomic Library
One other reason why you may not be able to find your gene of interest in
your library is that it contains within its sequence one or more sites for the