Cutting and joining DNA molecules - Principles of Gene Manipulation - page 41

in the two strands at each join were repaired in
vitro with DNA polymerase and DNA ligase so as to
produce covalent-ly closed circular molecules. The
recombinants were then transfected into susceptible
mammalian cells (see Chapter 10). Subsequently,
the homopolymer method, using either dA.dT or
dG.dC homopolymers was used extensively to con-
struct recombinant plasmids for cloning in E. coli . In
recent years, homopolymer tailing has been largely
replaced as a result of the availability of a much
wider range of restriction endonucleases and other
DNA-modifying enzymes. However, it is still impor-
tant for cDNA cloning (see p. 95 et seq .).

Incorporation of extra sequence at the
5 ′ end of a primer into amplified DNA

A PCR primer may be designed which, in addition
to the sequence required for hybridization with the
input DNA, includes an extra sequence at its 5 end.

The extra sequence does not participate in the first
hybridization step - only the 3

′

portion of the primer
hybridizes - but it subsequently becomes incorpo-
rated into the amplified DNA fragment (Fig. 3.13).
Because the extra sequence can be chosen at the
will of the experimenter, great flexibility is available
here.
A common application of this principle is the
incorporation of restriction sites at each end of the
amplified product. Figure 3.13 illustrates the addi-
tion of a Hin dIII site and an Eco RI site to the ends
of an amplified DNA fragment. In order to ensure
that the restriction sites are good substrates for
the restriction endonucleases, four nucleotides are
placed between the hexanucleotide restriction sites
and the extreme ends of the DNA. The incorporation
of these restriction sites provides one method for
cloning amplified DNA fragments (see below).

′

Joining polymerase chain
reaction (PCR) products

Many of the strategies for cloning DNA fragments do
not work well with PCR products. The reason for this
is that the polymerases used in the PCR have a ter-
minal transferase activity. For example, the Taq
polymerase adds a single 3 A overhang to each end

of the PCR product. Thus PCR products cannot be
blunt-end-ligated unless the ends are first polished
(blunted). A DNA polymerase like Klenow can be
used to fill in the ends. Alternatively, Pfu DNA poly-
merase can be used to remove extended bases with
its 3

′

Joining DNA molecules without
DNA ligase

exonuclease activity. However, even
when the PCR fragments are polished, blunt-end-
ligating them into a vector still may be very
inefficient. One solution to this problem is to use T/A
cloning (Mead et al. 1991). In this method, the PCR
fragment is ligated to a vector DNA molecule with a
single 3

′ to 5 ′

In all the cutting and joining reactions described
above, two separate protein components were
required: a site-specific endonuclease and a DNA lig-
ase. Shuman (1994) has described a novel approach
to the synthesis of recombinant molecules in which
a single enzyme, vaccinia DNA topoisomerase, both ′ deoxythymidylate extension (Fig. 3.12).

(a)

GG T G ANNNNNNNNNN - - - - -
- - - - -

C C A C T NNNNNNNNNN

(b) Hph I

Fig. 3.12 Cleavage of a vector DNA
molecule to generate single thymidylate
overhangs. (a) The recognition
sequence and cleavage point for the
restriction endonuclease Hph I.
(b) Sequences in the vector DNA which
result in desired overhangs after
cleavage with Hph I.

GGTGA ACCCGGGT T CGA
CCACT TGGGCCGAAGCT

- - - - - - - - - -
- - - - - - - - - -

AGAACCCGGGT TCACC

T C T TGGGCCCA AGTGG

Hph I

Hph I

GGTGA ACCCGGGT
CCACT TGGGCCC

CCCGGGT TCACC
TGGGCCCAAGTGG