namely a prokaryotic origin of DNA replication in the case of bacterial artificial chromosomes(BACs) or
a centromere(the region required for correct segregation of daughter chromosomes during mitosis and
meiosis) and two telomeres(the ends of the chromosome) in the case of YACs. BACs accept inserts of up to
about 150,000 bp and YACs can accept inserts of many hundreds or thousands of base pairs. Both types repli-
cate inside their host cells in exactly the same way as a natural chromosomes. This carries the disadvantage
that only one YAC molecule can exist per yeast cell. BACs are preferred nowadays over YACs because YACs
have problems with instability of the inserts and an unacceptably high occurrence of chimeric inserts
derived from more than one genomic region.
The choice of vector is dependent on the ease of screening, the transformation efficiency, the insert size
and the ease of isolating the DNA after cloning. If the average insert size is large, then less recombinants
are needed to obtain a representative recombinant library. Vector DNA use allows not only an original
cloning to be carried out, but also sub-cloning into more amenable fragments later in a project.
5.2.1.2 DNA Inserts. Often the source of the nucleic acid is a cDNA copy of messenger RNA (mRNA),
which has been generated using the enzyme reverse transcriptase. Sometimes the DNA of interest can be syn-
thesised chemically (Section 4.1). More often now it is the product of PCR (Section 5.2.2). Often, the
DNA to be cloned is inserted as a duplex into a restriction site of the vector DNA, after treatment of the vec-
tor with that restriction enzyme, by use of the enzyme DNA ligase (Section 5.3.5, Figure 5.2). Usually the
insert DNA is a restriction fragment with termini compatible with the vector ends. Sometimes oligonucleotide
linkersneed to be joined to the insert. These are self-complementary synthetic duplex oligonucleotides that
specify a recognition site for a restriction enzyme. Linkers are ligated to the fragment to be cloned and then
treated with the restriction enzyme, thus generating new termini, which are now identical or compatible for
joining to the cleaved vector. In all cases, the joined DNA is then transfected into a host cell line.
5.2.1.3 Identification of Clones. The rate-limiting step in classical cloning is often the identification
of the correct clone from a huge excess of other molecules. Typically, a vast number of visually identical
bacterial colonies or bacteriophage plaques are generated on the surface of agar in Petri dishes. To identify
the very few clones of interest, several approaches can be used. Often, a copy of the entire set of recombin-
ants is made by touching a nitrocellulose or nylon filter membrane to the agar surface. The ‘master’ copy
agar plate is stored away until the location of the required clone has been established on the filter copy.
Nucleic Acids in Biotechnology 171
Figure 5.2 Basic cloning procedure