John M. S. Bartlett.pdf - Bio-Nica.info

482 Horton, Raju, and Conti-Fine
other end. Together with the T-linkers using split NdeI and HindIII sites described here, a
T-linker that introduces a split SacI site, for example, would complete a set.
3. Potentially, other DNA sequences, such as a promoter for in vitro transcription, could be
split so that a functional site is completed at only one end of the product. If the majority
of the DNA sequence of such sites can be added by ligating a T-linker, this could provide
significant cost savings compared to synthesizing target-specific primers containing such
sequences.
4. Blunt-ended ligation of linkers has been used to add primer sequences to DNA fragments
(9,10), but the T-linker application presented here is novel as far as we can tell. The
T-linkers are more suitable for use with DNA fragments generated by PCR than bluntended
linkers, because of the nontemplate derived as added by the polymerase. Because
the sequences at the ends of PCR products can be easily manipulated by incorporating
changes in the primers, DNA sequences, such as restriction sites, can be split between
the T-linker and the PCR product. In general, this sort of site splitting is not practical
except with PCR-generated fragments. One exception is fragments tailed with a known
homopolymer, such as the poly A tail on eukaryotic mRNAs (11); such a linker is commercially
available (Novagen, Madison, WI). Because the T-linker is added as an inverted
repeat at the ends of the fragment, a single primer (TL-A) is used to reamplify after
ligation. Single-primer amplification systems (12) have an advantage in that a “primerdimer”
cannot be formed from one primer, because this would produce an unamplifiable
hairpin.
5. Because each product being cloned is subjected to an extra amplification, the overall
frequency of errors should be increased, although our experience shows that the increase
is not dramatic. In many cases, the risk of PCR errors is quite acceptable. For example, if
one is producing an enzyme or other protein with a measurable function, the clones can be
screened for activity. Deleterious mutations will thus be weeded out, and nondeleterious
mutations may not matter (the α3 subunit in our example will be used as a potential
ligand-binding protein, and clones will be screened on this basis). Similarly, if a sequence
is to be used as a hybridization probe, a low frequency of base substitutions is frequently
acceptable. In situations where no mutations are acceptable, clones must be screened
by careful sequencing.
6. The pH and the magnesium concentration of the PCR are different from those of the
ligation. Using a small ligation volume and a large PCR volume helps to correct the
conditions for the PCR. Alternatively, the pH of the bottom mix can be increased, and
the added magnesium reduced, so that the final pH and (Mg 2+ ) are correct after mixing.
This allows use of smaller volumes. The ability to make a quick, automated one-tube
ligation/reamplification reaction makes this setup intriguing. However, it is easier to
repeat parts of the experiment (such as the reamplification) if you have leftovers from
a separate ligation reaction.
Acknowledgments
This work was supported by research grants from the Muscular Dystrophy Association
of America and from the Council for Tobacco Research, by the NIH grant N52319,
and the NIDA Program Project grant DA05695. R. M. H. was the recipient of the
Robert G. Sampson Neuromuscular Disease Research Fellowship from the Muscular
Dystrophy Association.
References
1. Clark, J. M. (1988) Novel nontemplated nucleotide addition reactions catalyzed by
procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16, 9677–9686.