Gene Cloning

Gene Identification and DNA Libraries 111
Because the S. cerevisiae genome is larger, than the bacterial genomes we
have considered so far, you would need 13,970 clones in your library to have
a 99% chance of a particular gene being present.
Q4.5. How many clones of E. coli would you need to have a 99.9% chance
of cloning a particular gene?
A4.5. You would need 8010 clones to have a 99.9% chance of cloning your
gene. This is nearly twice as many clones and you have only increased your
chances by 0.9%. This is sometimes referred to as the law of diminishing
returns; you have to decide whether increasing the number of clones is
worthwhile.
Q4.6. Using a partial Sau3AI digest you have isolated DNA fragments from
Pseudomonas with an average size of 10 kb and constructed a genomic
library. How many clones would you need to have a probability of 0.99 of
finding the gene of interest?
A4.6. Using the formula with f = 10, g = 6300 and P = 0.99,
ln (1 – 0.99) ln 0.01
N = =
ln (1 – 10/6300) ln 0.9984
– 4.61
= = 2899.
–1.58 85
× 10 –3
With these larger sized fragments you would only need 2900 clones compared
with over 7000 for a library made with 4 kb fragments.
Q4.7. Using a Sau3AI partial digest presents a problem when you come to
cutting your cloning vector. Can you see what the problem is? Can you
think of a cloning strategy which would overcome this problem? Hints.
How many times would you expect Sau3AI to cut pUC18? Is this a problem?
Is there a restriction enzyme with sticky ends, which are compatible with
those produced by Sau3AI, with a unique site in pUC18?
A4.7. Four base cutters like Sau3AI cut on average every 256 bp so it is likely
that any cloning vector will be cut into many pieces rather than being
opened up at a single site. Refer to Figure 3.10; you can cut the vector with
BamHI or BglII and ligate the Sau3AI fragments into the site. Many commonly
used cloning vectors have a unique restriction site for at least one of
these enzymes, both of which produce sticky ends which are compatible with
those from Sau3AI.
Q4.8. What factors may cause some clones to be less well represented in
the library than others?