218 MaundersThree types of units are defined commonly. First, a Weiss unit (17)
catalyzes the exchange of 1 nmol of 32p from inorganic pyrophosphate
to ATP in 20 min at 37°C, 0.015 Weiss units ligate 50 % of the HindlII
fragments from 5 lag ~, DNA in 30 min at 16°C. Second, the Modrich-
Lehman unit, based on the exonuclease resistance assay (3 7), is equiva-
lent to 5 Weiss units. Finally, cohesive end units are functionally defined
by various commercial suppliers, and are generally much smaller than
Weiss units and difficult to relate quantitatively.
- Experimental Procedures
3.1. Uses of DNA Ligases
The most widespread use of DNA ligase is in the construction of
recombinant DNA molecules. This may be necessary for the cloning
of cDNA or genomic fragments for construction of libraries, or for
mapping, sequencing, or use as probes. The use of ligase in the inverse
polymerase chain reaction facilitates the cloning of segments of geno-
mic DNA some distance from known sequences (41). DNA ligase may
also be used in the assembly of genes from DNA fragments and syn-
thetic oligomers.
Other uses of DNA ligase include the detection of nicked DNA by
the release of AMP (38), nearest neighbor analysis following kinasing
(42,43), use in mutagenesis (44), and the making of affinity columns
by attaching DNA to solid matrices. In most cases, the enzyme of
choice is T4 DNA ligase. E. coli ligase is not widely used, because it
is inefficient with blunt ends. However, E. coli DNA ligase is used for
cDNA cloning by replacement synthesis (45) where a virtue is made
of its inability to ligate RNA to DNA to form spurious products.
Examples of specific ligation protocols may be found in Gaastra
and Hansen (46). The following details apply to the use of T4 DNA
ligase, except where specific reference is made to the E. coli enzyme.
3.2. Storage and Stability
T4 DNA ligase is usually supplied at a concentration of 1-5 U/I.tL.
It can be diluted in reaction buffer immediately prior to ligation, but
for longer term storage, a specific storage buffer should be used.
Storage buffers usually contain:
- 10-20 mM Tris-HCl (or potassium phosphate), pH 7.4-7.6
- 50-60 mM KC1