Genetic Manipulation 217Figure 9.1 Restriction enzymes
over a very limited time period with an exonuclease which digests the end of
the DNA and followed by further digestion with another nuclease which tidies
up the ends to produce flush ends. There are other restriction nucleases which
recognise a site in DNA but cut at some distance from it, but these are rarely of
any value in cloning procedures.
Preparation of the vector is dictated by the type of end prepared for the insert
DNA: flush or ‘sticky’. If it is flush, it does not much matter how that was
achieved so long as the vector receiving it is also flush, but if it is sticky, the
appropriate sticky end must be prepared on the vector by a suitable restric-
tion endonuclease. There are many methods of DNA and vector preparation all
of which have their advantages and disadvantages and, although interesting in
themselves, are beyond the scope of this book.
Having prepared the ends, the next step is to stick the pieces together. The
prepared insert, or ‘foreign’ DNA is incubated with the prepared vector in an
aqueous solution containing various salts required by the enzymes, and ligase
which is an enzyme, the function of which is to make the bond between the free
phosphate on a nucleotide base and the neighbouring ribose sugar, thus ‘repairing’
the DNA to make a complete covalently linked chain. This recombinant DNA
molecule may be transferred into a cell where it undergoes replication in the
usual way. If the DNA is not viral, introduction will be by direct entry through
the cell membrane achieved by any one of a number of standard techniques all