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may need to be repaired to make them flush-ended. This can be achieved by using a

modified DNA polymerase termed Klenow polymerase. This is prepared by cleavage of

DNA polymerase with subtilisin, giving a large enzyme fragment which has no 5^0 to 3^0

exonuclease activity, but which still acts as a 5^0 to 3^0 polymerase. This will fill in any

recessed 3^0 ends on the sheared DNA using the appropriate dNTPs.

The mixture of DNA fragments is then ligated with a vector, and subsequently cloned.

If enough clones are produced there will be a very high chance that any particular

DNA fragment such as a gene will be present in at least one of the clones. To keep the

number of clones to a manageable size, fragments about 10kb in length are needed for

prokaryotic libraries, but the length must be increased to about 40kb for mammalian

libraries. It is possible to calculate the number of clones that must be present in a gene

library to give a probability of obtaining a particular DNA sequence. This formula is:

N¼lnð 1 PÞ

lnð 1 fÞ

whereNis the number of recombinants,Pis the probability andfis the fraction of the

genome in one insert. Thus for theE. coliDNA chromosome of 5 106 bp and with an

insert size of 20 kb the number of clones needed (N) would be 1 103 with a

probability of 0.99.

6.2.5 cDNA libraries

There may be several thousand different proteins being produced in a cell at any one

time, all of which have associated mRNA molecules. To identify any one of those

(a) E E

(b)

EE

EE E E

Fig. 6.4Comparison of (a) partial and (b) complete digestion of DNA molecules at restriction enzyme sites (E).

200 Recombinant DNA and genetic analysis