76 Gerard and D'Alessio2.4. Catalytic Properties
There are two major difficulties with RT-catalyzed eDNA synthe-
sis: (1) mass yields of eDNA transcripts generally do not exceed 50%,
and (2) many eDNA transcripts are less than full length. When AMV
RT was first used to synthesize eDNA, these difficulties were caused
in part by RNase contaminants in AMV RT preparations. However,
commercial preparations ofAMV RT are now much cleaner. The high
level of expression of cloned M-MLV RT and M-MLV H- RT in E. coIi
has made it possible to eliminate detectable RNase contamination
from enzyme preparations. However, results still fall short of the ulti-
mate objective of synthesizing one full-length eDNA copy of every
mRNA in a population, and this is owing in part to the catalytic prop-
erties of all RTs.
2.4.1. DNA Polymerase
RT resembles other template-directed DNA polymerases. It catalyzes
the synthesis of DNA in the presence of a preformed primer with a 3'-OH
hydrogen bonded to a template polynucleotide, a divalent metal ion, and
a mixture of all four dNTPs (21). In contrast to most other DNA poly-
merases, RT can use either RNA or DNA to prime DNA synthesis from
an RNA or DNA template. As a DNA polymerase, RT displays low
processivity (22,23) and elongates DNA chains slowly (23). RT tends to
pause at certain sequence and secondary structure-specific domains in
RNA (7). As discussed below (see Section 2.4.2.), such pausing can pre-
vent an mRNA molecule from being copied all the way to its 5' end.
There is no endogenous 3'-5' exonuclease editing function associ-
ated with RT (21). In spite of this, the error rate of cloned M-MLV RT
is low (1 mismatched residue in 30,000 incorporated; ref. 24). When the
RNase H activity of cloned M-MLV RT was deleted to produce M-MLV
H- RT, the error rate did not change (19).
2.4.2. RNase H
The RNase H activity of native RT specifically degrades the RNA
strand of RNA • DNA (21) and can act independently of the RT DNA
polymerase activity (25). The initiation of cDNA synthesis depends
on the hybridization ofa DNA primer to mRNA, usually at the poly(A)
tail. When this occurs, the hybrid is not only a priming site for the
polymerase activity of RT, but is also a substrate for the RT RNase H