Ligases 221
of the DNA fragment to be linked. The effective concentration of the
DNA can be increased by the use of condensing agents or volume exclud-
ers, such as polyethylene glycol (PEG), Ficoll, and hexamine cobalt
chloride (53), which accelerate the rate of ligation by up to 1000-fold,
and permit ligation at lower absolute DNA and enzyme concentra-
tions. Condensing agents alter the distribution of the products and
suppress intramolecular reaction (recircularization), most products
being linear multimers.
PEG8000 gives maximum stimulation of ligation when present at a
concentration of 15 %, this effect being achieved at a concentration of
0.5 mM ATP and 5 mM MgC12. Even slight increases in the ATP or
decreases in the magnesium ion concentrations greatly diminish the
effect of PEG (54). PEG enhances the ligation of blunt-ended oligo-
mers as short as 8 bp and also simulates ligation of cohesive ends by
10-100-fold.
Hexamine cobalt chloride stimulation depends on the concentration
used, 1.0-1.5 laM having maximal effect. Blunt-end ligation is stimu-
lated 50-fold, but cohesive-end ligation only fivefold. Ligation is pos-
sible in the presence of monovalent cations (e.g., 30 mM KC1), but the
products are then largely recircularized. This reagent does not signifi-
cantly increase the ligation of short oligomers.
T4 RNA ligase will stimulate T4 DNA ligase activity (24), but is not
widely used since PEG is more efficient and inexpensive. RNA ligase
cannot perform blunt-end DNA ligations itself, nor will it activate the
E. coli enzyme.
3.5. Ligation Products
The products of ligation depend largely on the nature of the DNA
substrates employed. Removal of the 5'-phosphate groups from one of
the substrate molecules (e.g., a vector) by alkaline phosphatase treat-
ment will prevent self-ligation and favor the formation of recombinant
products. The use of DNA molecules carrying different cohesive ter-
mini at each end (asymmetric cloning) also prevents self-ligation and
offers control over the relative orientation of the DNA fragments in the
resultant products.
The products obtained also depend on the absolute concentrations
and ratios of the DNA substrates present in the mixture. Low DNA
concentrations favor intramolecular reaction (recircularization), and