cDNA Synthesis with M-MLV RT 81
The cloned M-MLV RTs are purified free of detectable RNase con-
tamination. If precautions are taken to avoid adventitious introduction
of RNase contaminants into reaction mixtures, RNase inhibitors are
not necessary, and addition of such inhibitors can increase the chances
of introducing contaminants into a reaction mixture.
- 7.6. Actinomycin D
Actinomycin D at 50-100 ~tg/mL inhibits the synthesis of double-
stranded from single-stranded DNA (45). It is added during first-strand
cDNA synthesis to inhibit hairpin primed double-stranded DNA syn-
thesis. It probably interacts with 3' single-stranded cDNA ends in such
a way that DNA • DNA hybridization needed for hairpin formation is
inhibited (46). In first-strand cDNA reactions catalyzed by M-MLV
RT, the proportion ofcDNA molecules generated with exposed 3' single-
stranded ends can be controlled to a large extent in the absence of
actinomycin D by adjusting the ratio of enzyme to mRNA in the reac-
tion. No actinomycin D is required in reactions catalyzed by M-MLV
H- RT, since first-strand cDNA product remains hybridized to intact
mRNA template, and hairpin primed DNA synthesis cannot occur.
- Inhibitors
Glycerol concentrations as high as 19% can be tolerated in RT reac-
tion mixtures (Gerard, G., unpublished). Both M-MLV RTs are inhib-
ited by phosphate, pyrophosphate, and polyamines.
- 7.8. Reducing Agent
Both M-MLV RTs require dithiothreitol (DTT) for optimal activity.
A minimum concentration of 5 mM is necessary, although l0 mM are
optimal. Aqueous, unbuffered solutions of 0.1M DTT are stable at
20°C, even after repeated freeze-thaw cycles. However, DTT is oxi-
dized quite rapidly in buffered solutions above pH 7.5, so that reaction
buffers that include DTT should be prepared freshly and discarded
after use. No data are available on the effect of [~-mercaptoethanol on
M-MLV RTs.
2.7.9. Temperature
When either M-MLV RT or M-MLV H- RT is used to synthesize
first-strand cDNA, the cDNA libraries with the largest average insert
size result when first-strand cDNA synthesis is performed at 37°C.
